Proteins

ABSTRACT

The present invention relates to a method of producing a variant lipid acyltransferase enzyme comprising: (a) selecting a parent enzyme which is a lipid acyltransferase enzyme characterised in that the enzyme comprises the amino acid sequence motif GDSX, wherein X is one or more of the following amino acid residues L, A, V, I, F, Y, H, Q, T N, M or S; (b) modifying one or more amino acids to produce a variant lipid acyltransferase; (c) testing the variant lipid acyltransferase for activity on a galactolipid substrate, and optionally a phospholipid substrate and/or optionally a triglyceride substrate; (d) selecting a variant enzyme with an enhanced activity towards galactolipids compared with the parent enzyme; and optionally (e) preparing a quantity of the variant enzyme. The present invention further relates to variant lipid acyltransferase enzyme characterised in that the enzyme comprises the amino acid sequence motif GDSX, wherein X is one or more of the following amino acid residues L, A, V, I, F, Y, H, Q, T N, M or S, and wherein the variant enzyme comprises one or more amino acid modifications compared with a parent sequence at any one or more of the following amino acid residues when aligned to SEQ ID No. 2: Ser3, Leu17, Ala114, Trp111, Tyr117, Leu118, Pro156, Gly159, Gln160, Asn161, Pro162, Ser163, Ala164, Arg165, Ser166, Gln167, Lys168, Val169, Val170, Glu171, Ala172, Tyr179, Gln182, Lys187, His180, Asn181, Met209, Leu210, Arg211, Asn215, Lys284, Met285, Gln289, Val290, Ala309, Ser310, Lys22, Met23, Gly40, Asn80, Pro81, Lys82, Val112, Asn87, Asn88, −318.

REFERENCE TO RELATED APPLICATIONS

This application claims priority from United Kingdom Application Number GB 0330016.7 filed on 24 Dec. 2003, International Patent Application Number PCT/IB2004/000655 filed on 15 Jan. 2004 and United Kingdom Application Number GB 0415999.2 filed on 16 Jul. 2004.

Reference is also made to the following related applications: U.S. application Ser. No. 09/750,990 filed on 20 Jul. 1999; U.S. application Ser. No. 10/409,391 and U.S. Application Ser. No. 60/489,441 filed on 23 Jul. 2003.

Each of these applications and each of the documents cited in each of these applications (“application cited documents”), and each document referenced or cited in the application cited documents, either in the text or during the prosecution of those applications, as well as all arguments in support of patentability advanced during such prosecution, are hereby incorporated herein by reference. Various documents are also cited in this text (“herein cited documents”). Each of the herein cited documents, and each document cited or referenced in the herein cited documents, is hereby incorporated herein by reference.

FIELD OF INVENTION

The present invention relates to methods of producing variant enzymes. The present invention further relates to novel variant enzymes and to the use of these novel variant enzymes.

TECHNICAL BACKGROUND

Lipid:cholesterol acyltransferase enzymes have been known for some time (see for example Buckley—Biochemistry 1983, 22, 5490-5493). In particular, glycerophospholipid:cholesterol acyl transferases (GCATs) have been found, which like the plant and/or mammalian lecithin:cholesterol acyltransferases (LCATs), will catalyse fatty acid transfer between phosphatidylcholine and cholesterol.

Upton and Buckley (TIBS 20, May 1995, p 178-179) and Brumlik and Buckley (J. of Bacteriology Apr. 1996, p 2060-2064) teach a lipase/acyltransferase from Aeromonas hydrophila which has the ability to carry out acyl transfer to alcohol receptors in aqueous media.

A putative substrate binding domain and active site of the A. hydrophila acyltransferase have been identified (see for example Thornton et al 1988 Biochem. et Biophys. Acta. 959, 153-159 and Hilton & Buckley 1991 J. Biol. Chem. 266, 997-1000) for this enzyme.

Buckley et al (J. Bacteriol 1996, 178(7) 2060-4) taught that Ser16, Asp116 and His291 are essential amino acids which must be retained for enzyme activity to be maintained.

Robertson et al (J. Biol. Chem. 1994, 269, 2146-50) taught some specific mutations, namely Y226F, Y230F, Y30F, F13S, S18G, S18V, of the A. hydrophila acyltransferase, none of which are encompassed by the present invention.

SUMMARY ASPECTS OF THE PRESENT INVENTION

The present invention is predicated upon the finding of specific variants of a GDSx containing lipid acyltransferase enzyme, which variants have an increased hydrolytic activity and/or transferase activity compared with a parent enzyme. In particular, the variants according to the present invention have an enhanced hydrolytic activity towards galactolipids and/or an enhanced transferase activity using galactolipid as an acyl donor as compared with a parent enzyme. The variants according to the present invention may additionally have an enhanced ratio of activity towards galactolipids to phospholipids and/or towards galactolipids to triacylglyerides compared with a parent enzyme.

According to a first aspect the present invention provides a method of producing a variant lipid acyltransferase enzyme comprising: (a) selecting a parent enzyme which is a lipid acyltransferase enzyme characterised in that the enzyme comprises the amino acid sequence motif GDSX, wherein X is one or more of the following amino acid residues L, A, V, I, F, Y, H, Q, T N, M or S; (b) modifying one or more amino acids to produce a variant lipid acyltransferase; (c) testing the variant lipid acyltransferase for activity on a galactolipid substrate, and optionally a phospholipid substrate and/or optionally a triglyceride substrate; (d) selecting a variant enzyme with an enhanced activity towards galactolipids compared with the parent enzyme; and optionally (e) preparing a quantity of the variant enzyme.

In another aspect the present invention provides a variant lipid acyltransferase enzyme characterised in that the enzyme comprises the amino acid sequence motif GDSX, wherein X is one or more of the following amino acid residues L, A, V, I, F, Y, H, Q, T N, M or S, and wherein the variant enzyme comprises one or more amino acid modifications compared with a parent sequence at any one or more of the following amino acid residues when aligned to SEQ ID No. 2: Ser3, Leu17, Ala114, Trp111, Tyr117, Leu118, Pro156, Gly159, Gln160, Asn161, Pro162, Ser163, Ala164, Arg165, Ser166, Gln167, Lys168, Val169, Val170, Glu171, Ala172, Tyr179, Gln182, Lys187, His180, Asn181, Met209, Leu210, Arg211, Asn215, Lys284, Met285, Gln289, Val290, Ala309, Ser310, Lys22, Met23, Gly40, Asn80, Pro81, Lys82, Val112, Asn87, Asn88, −318.

In a further aspect the present invention provides a variant lipid acyltransferase enzyme characterised in that the enzyme comprises the amino acid sequence motif GDSX, wherein X is one or more of the following amino acid residues L, A, V, I, F, Y, H, Q, T N, M or S, and wherein the variant enzyme comprises one or more amino acid modifications compared with a parent sequence at any one or more of the following amino acid residues identified by said parent sequence being structurally aligned with the structural model of P10480 defined herein, which is preferably obtained by structural alignment of P10480 crystal structure coordinates with 1IVN.PDB and/or 1DEO.PDB as taught herein: Ser3, Leu17, Ala114, Trp111, Tyr117, Leu118, Pro156, Gly159, Gln160, Asn161, Pro162, Ser163, Ala164, Arg165, Ser166, Gln167, Lys168, Val169, Val170, Glu171, Ala172, Tyr179, Gln182, Lys187, His180, Asn181, Met209, Leu210, Arg211, Asn215, Lys284, Met285, Gln289, Val290, Ala309, Ser310, Lys22, Met23, Gly40, Asn80, Pro81, Lys82, Val112, Asn87, Asn88, −318.

The present invention yet further provides a variant lipid acyltransferase enzyme characterised in that the enzyme comprises the amino acid sequence motif GDSX, wherein X is one or more of the following amino acid residues L, A, V, I, F, Y, H, Q, T N, M or S, and wherein the variant enzyme comprises one or more amino acid modifications compared with a parent sequence at any one or more of the following amino acid residues identified when said parent sequence is aligned to the pfam consensus sequence (SEQ ID No. 1) and modified according to a structural model of P10480 to ensure best fit overlap (see FIG. 55) as taught herein: Ala114, Trp111, Tyr117, Pro156, Tyr179, Gln182, Lys187, His180, Asn181, Met209, Leu210, Arg211, Asn215, Met285, Gln289, Val290, Asn80, Pro81, Lys82.

According to a further aspect the present invention provides a variant lipid acyltransferase enzyme wherein the variant enzyme comprises an amino acid sequence, which amino acid sequence is shown as SEQ ID No. 2, SEQ ID No. 3, SEQ ID No. 4, SEQ ID No. 5, SEQ ID No. 6, SEQ ID No. 12, SEQ ID No. 14, SEQ ID No. 16, SEQ ID No. 18, SEQ ID No. 20, SEQ ID No. 22, SEQ ID No. 24, SEQ ID No. 26, SEQ ID No. 28, SEQ ID No. 30, SEQ ID No. 33, SEQ ID No. 34, SEQ ID No. 36, SEQ ID No. 37, SEQ ID No. 39, SEQ ID No. 41, SEQ ID No. 43 or SEQ ID No. 45 except for one or more amino acid modifications at any one or more of the following amino acid residues identified by sequence alignment with SEQ ID No. 2: Ser3, Leu17, Ala114, Trp111, Tyr117, Leu118, Pro156, Gly159, Gln160, Asn161, Pro162, Ser163, Ala164, Arg165, Ser166, Gln167, Lys168, Val169, Val170, Glu171, Ala172, Tyr179, Gln182, Lys187, His180, Asn181, Met209, Leu210, Arg211, Asn215, Lys284, Met285, Gln289, Val290, Ala309, Ser310, Lys22, Met23, Gly40, Asn80, Pro81, Lys82, Val112, Asn87, Asn88, −318.

In a further aspect the present invention provides a variant lipid acyltransferase enzyme wherein the variant enzyme comprises an amino acid sequence, which amino acid sequence is shown as SEQ ID No. 2, SEQ ID No. 3, SEQ ID No. 4, SEQ ID No. 5, SEQ ID No. 6, SEQ ID No. 12, SEQ ID No. 14, SEQ ID No. 16, SEQ ID No. 18, SEQ ID No. 20, SEQ ID No. 22, SEQ ID No. 24, SEQ ID No. 26, SEQ ID No. 28, SEQ ID No. 30, SEQ ID No. 33, SEQ ID No. 34, SEQ ID No. 36, SEQ ID No. 37, SEQ ID No. 39, SEQ ID No. 41, SEQ ID No. 43 or SEQ ID No. 45 except for one or more amino acid modifications at any one or more of the following amino acid residues identified by said parent sequence being structurally aligned with the structural model of P10480 defined herein, which is preferably obtained by structural alignment of P10480 crystal structure coordinates with 1IVN.PDB and/or 1DEO.PDB as taught herein: Ser3, Leu17, Ala114, Trp111, Tyr117, Leu118, Pro156, Gly159, Gln160, Asn161, Pro162, Ser163, Ala164, Arg165, Ser166, Gln167, Lys168, Val169, Val170, Glu171, Ala172, Tyr179, Gln182, Lys187, His180, Asn181, Met209, Leu210, Arg211, Asn215, Lys284, Met285, Gln289, Val290, Ala309, Ser310, Lys22, Met23, Gly40, Asn80, Pro81, Lys82, Val112, Asn87, Asn88, −318.

According to a further aspect the present invention provides a variant lipid acyltransferase enzyme wherein the variant enzyme comprises an amino acid sequence, which amino acid sequence is shown as SEQ ID No. 2, SEQ ID No. 3, SEQ ID No. 4, SEQ ID No. 5, SEQ ID No. 6, SEQ ID No. 12, SEQ ID No. 14, SEQ ID No. 16, SEQ ID No. 18, SEQ ID No. 20, SEQ ID No. 22, SEQ ID No. 24, SEQ ID No. 26, SEQ ID No. 28, SEQ ID No. 30, SEQ ID No. 33, SEQ ID No. 34, SEQ ID No. 36, SEQ ID No. 37, SEQ ID No. 39, SEQ ID No. 41, SEQ ID No. 43 or SEQ ID No. 45 except for one or more amino acid modifications at any one or more of the following amino acid residues identified when said parent sequence is aligned to the pfam consensus sequence (SEQ ID No. 1) and modified according to a structural model of P10480 to ensure best fit overlap (see FIG. 55) as taught herein: Ala114, Trp111, Tyr117, Pro156, Tyr179, Gln182, Lys187, His180, Asn181, Met209, Leu210, Arg211, Asn215, Met285, Gln289, Val290, Asn80, Pro81, Lys82.

The present invention yet further provides the use of a variant lipolytic enzyme according to the present invention or obtained by a method according to the present invention in a substrate (preferably a foodstuff) for preparing a lyso-glycolipid, for example digalactosyl monoglyceride (DGMG) or monogalactosyl monoglyceride (MGMG) by treatment of a glycolipid (e.g. digalactosyl diglyceride (DGDG) or monogalactosyl diglyceride (MGDG)) with the variant lipolytic enzyme according to the present invention or obtained by a method according to the present invention to produce the partial hydrolysis product, i.e. the lyso-glycolipid.

In a further aspect, the present invention provides the use of a variant lipolytic enzyme according to the present invention or obtained by a method according to the present invention in a substrate (preferably a foodstuff) for preparing a lyso-phospholipid, for example lysolecithin, by treatment of a phospholipid (e.g. lecithin) with the variant lipolytic enzyme according to the present invention or obtained by a method according to the present invention to produce a partial hydrolysis product, i.e a lyso-phospholipid.

In one aspect the present invention relates to a method of preparing a foodstuff the method comprising adding a variant lipolytic enzyme according to the present invention or obtained by a method according to the present invention to one or more ingredients of the foodstuff.

Another aspect of the present invention relates to a method of preparing a baked product from a dough, the method comprising adding a variant lipolytic enzyme according to the present invention or obtained by a method according to the present invention to the dough.

In another aspect of the present invention there is provided the use of a variant lipolytic enzyme according to the present invention or obtained by a method according to the present invention in a process of treating egg or egg-based products to produce lysophospholipids.

A further aspect of the present invention provides a process of enzymatic degumming of vegetable or edible oils, comprising treating the edible or vegetable oil with a variant lipolytic enzyme according to the present invention or obtained by a method according to the present invention so as to hydrolyse a major part of the polar lipids (e.g. phospholipid and/or glycolipid).

In another aspect the present invention provides the use of a variant lipolytic enzyme according to the present invention or obtained by a method according to the present invention in a process comprising treatment of a phospholipid so as to hydrolyse fatty acyl groups.

In another aspect the present invention provides the use of a variant lipolytic enzyme according to the present invention or obtained by a method according to the present invention in a process for reducing the content of a phospholipid in an edible oil, comprising treating the oil with said variant lipolytic enzyme so as to hydrolyse a major part of the phospholipid, and separating an aqueous phase containing the hydrolysed phospholipid from the oil.

There is also provided a method of preparing a variant lipolytic enzyme according to the present invention or obtained by a method according to the present invention, the method comprising transforming a host cell with a recombinant nucleic acid comprising a nucleotide sequence coding for said variant lipolytic enzyme, the host cell being capable of expressing the nucleotide sequence coding for the polypeptide of the lipolytic enzyme, cultivating the transformed host cell under conditions where the nucleic acid is expressed and harvesting the variant lipolytic enzyme.

In a further aspect the present invention relates to the use of a variant lipolytic enzyme according to the present invention or obtained by a method according to the present invention in the bioconversion of polar lipids (preferably glycolipids) to make high value products, such as carbohydrate esters and/or protein esters and/or protein subunit esters and/or a hydroxy acid ester.

The present invention yet further relates to an immobilised variant lipolytic enzyme according to the present invention or obtained by a method according to the present invention.

Aspects of the present invention are presented in the claims and in the following commentary.

Other aspects concerning the nucleotide sequences which can be used in the present invention include: a construct comprising the sequences of the present invention; a vector comprising the sequences for use in the present invention; a plasmid comprising the sequences for use in the present invention; a transformed cell comprising the sequences for use in the present invention; a transformed tissue comprising the sequences for use in the present invention; a transformed organ comprising the sequences for use in the present invention; a transformed host comprising the sequences for use in the present invention; a transformed organism comprising the sequences for use in the present invention. The present invention also encompasses methods of expressing the nucleotide sequence for use in the present invention using the same, such as expression in a host cell; including methods for transferring same. The present invention further encompasses methods of isolating the nucleotide sequence, such as isolating from a host cell.

Other aspects concerning the amino acid sequence for use in the present invention include: a construct encoding the amino acid sequences for use in the present invention; a vector encoding the amino acid sequences for use in the present invention; a plasmid encoding the amino acid sequences for use in the present invention; a transformed cell expressing the amino acid sequences for use in the present invention; a transformed tissue expressing the amino acid sequences for use in the present invention; a transformed organ expressing the amino acid sequences for use in the present invention; a transformed host expressing the amino acid sequences for use in the present invention; a transformed organism expressing the amino acid sequences for use in the present invention. The present invention also encompasses methods of purifying the amino acid sequence for use in the present invention using the same, such as expression in a host cell; including methods of transferring same, and then purifying said sequence.

For the ease of reference, these and further aspects of the present invention are now discussed under appropriate section headings. However, the teachings under each section are not necessarily limited to each particular section.

DETAILED ASPECTS OF THE PRESENT INVENTION

The variant lipid acyltransferase enzyme according to the present invention may in addition (or alternatively) to the modifications taught above, may comprise one of the following amino acid modifications at Ser18: S18A, L, M, F, W, K, Q, E, P, I, C, Y, H, R, N, D, T.

The variant lipid acyltransferase enzyme according to the present invention may in addition (or alternatively) to the modifications taught above, may comprise one of the following amino acid modifications at Y30: Y30A, G, L, M, W, K, Q, S, E, P, V, I, C, H, R, N, D, T.

The variant lipid acyltransferase enzyme according to the present invention may in addition (or alternatively) to the modifications taught above, may comprise one of the following amino acid modifications at Y230: Y230A, G, L, M, W, K, Q, S, E, P, V, I, C, H, R, N, D, T.

Preferably, the parent lipid acyltransferase enzyme comprises any one of the following amino acid sequences: SEQ ID No. 2, SEQ ID No. 3, SEQ ID No. 4, SEQ ID No. 5, SEQ ID No. 6, SEQ ID No. 12, SEQ ID No. 14, SEQ ID No. 16, SEQ ID No. 18, SEQ ID No. 20, SEQ ID No. 22, SEQ ID No. 24, SEQ ID No. 26, SEQ ID No. 28, SEQ ID No. 30, SEQ ID No. 33, SEQ ID No. 34, SEQ ID No. 36, SEQ ID No. 37, SEQ ID No. 39, SEQ ID No. 41, SEQ ID No. 43 or SEQ ID No. 45 or an amino acid sequence which has 75% or more identity with any one of the sequences shown as SEQ ID No. 2, SEQ ID No. 3, SEQ ID No. 4, SEQ ID No. 5, SEQ ID No. 6, SEQ ID No. 12, SEQ ID No. 14, SEQ ID No. 16, SEQ ID No. 18, SEQ ID No. 20, SEQ ID No. 22, SEQ ID No. 24, SEQ ID No. 26, SEQ ID No. 28, SEQ ID No. 30, SEQ ID No. 33, SEQ ID No. 34, SEQ ID No. 36, SEQ ID No. 37, SEQ ID No. 39, SEQ ID No. 41, SEQ ID No. 43 or SEQ ID No. 45.

Suitably, the parent lipid acyltransferase enzyme according to the present invention comprises an amino acid sequence which has at least 80%, preferably at least 85%, more preferably at least 90%, more preferably at least 95%, more at least 98% homology with any one of the sequences shown as SEQ ID No. 2, SEQ ID No. 3, SEQ ID No. 4, SEQ ID No. 5, SEQ ID No. 6, SEQ ID No. 12, SEQ ID No. 14, SEQ ID No. 16, SEQ ID No. 18, SEQ ID No. 20, SEQ ID No. 22, SEQ ID No. 24, SEQ ID No. 26, SEQ ID No. 28, SEQ ID No. 30, SEQ ID No. 33, SEQ ID No. 34, SEQ ID No. 36, SEQ ID No. 37, SEQ ID No. 39, SEQ ID No. 41, SEQ ID No. 43 or SEQ ID No. 45.

Suitably, the parent lipid acyltransferase enzyme may be encoded by any one of the following nucleotide sequences: SEQ ID No. 7, SEQ ID No. 8, SEQ ID No. 9, SEQ ID No. 10, SEQ ID No. 11, SEQ ID No. 13, SEQ ID No. 15, SEQ ID No. 17, SEQ ID No. 19, SEQ ID No. 21, SEQ ID No. 23, SEQ ID No. 25, SEQ ID No.27, SEQ ID No. 29, SEQ ID No. 31, SEQ ID No. 32, SEQ ID No. 35, SEQ ID No. 38, SEQ ID No. 40, SEQ ID No. 42, SEQ ID No. 44 or SEQ ID No. 46 or a nucleotide sequence which has at least 75% or more identity with any one of the sequences shown as SEQ ID No. 7, SEQ ID No. 8, SEQ ID No. 9, SEQ ID No. 10, SEQ ID No. 11, SEQ ID No. 13, SEQ ID No. 15, SEQ ID No. 17, SEQ ID No. 19, SEQ ID No. 21, SEQ ID No. 23, SEQ ID No. 25, SEQ ID No.27, SEQ ID No. 29, SEQ ID No. 31, SEQ ID No. 32, SEQ ID No. 35, SEQ ID No. 38, SEQ ID No. 40, SEQ ID No. 42, SEQ ID No. 44 or SEQ ID No. 46.

Suitably, the nucleotide sequence may have 80% or more, preferably 90% or more, more preferably 95% or more, even more preferably 98% or more identity with any one of the sequences shown as SEQ ID No. 7, SEQ ID No. 8, SEQ ID No. 9, SEQ ID No. 10, SEQ ID No. 11, SEQ ID No. 13, SEQ ID No. 15, SEQ ID No. 17, SEQ ID No. 19, SEQ ID No. 21, SEQ ID No. 23, SEQ ID No. 25, SEQ ID No.27, SEQ ID No. 29, SEQ ID No. 31, SEQ ID No. 32, SEQ ID No. 35, SEQ ID No. 38, SEQ ID No. 40, SEQ ID No. 42, SEQ ID No. 44 or SEQ ID No. 46.

Preferably, the parent enzyme is modified at one or more of the following amino acid residues Ser3, Leu17, Ala114, Trp111, Tyr117, Leu118, Pro156, Gly159, Gln160, Asn161, Pro162, Ser163, Ala164, Arg165, Ser166, Gln167, Lys168, Val169, Val170, Glu171, Ala172, Tyr179, Gln182, Lys187, His180, Asn181, Met209, Leu210, Arg211, Asn215, Lys284, Met285, Gln289, Val290, Ala309, Ser310, Lys22, Met23, Gly40, Asn80, Pro81, Lys82, Val112, Asn87, Asn88 when aligned to the reference sequence (SEQ ID No. 2) or structurally aligned to the structural model of P10480, or aligned to the pfam consensus sequence and modified according to the structural model of P10480.

Suitably the variant enzyme may have an enhanced ratio of activity on galactolipids to either phospholipids and/or triglycerides when compared with the parent enzyme.

The term “enhanced activity towards galactolipids” means the enzyme has an enhanced (i.e. higher) hydrolytic activity towards galactolipids and/or an enhanced (i.e. higher) transferase activity wherein the lipid acyl donor is a galactolipid.

The term “modifying” as used herein means adding, substituting and/or deleting. Preferably the term “modifying” means “substituting”.

For the avoidance of doubt, when an amino acid is substituted in the parent enzyme it is preferably substituted with an amino acid which is different from that originally found at that position in the parent enzyme. In other words, the term “substitution” is not intended to cover the replacement of an amino acid with the same amino acid.

Preferably, the parent enzyme is an enzyme which comprises the amino acid sequence shown as SEQ ID No. 2 and/or SEQ ID No. 28.

Preferably, the variant enzyme is an enzyme which comprises an amino acid sequence, which amino acid sequence is shown as SEQ ID No. 2 except for one or more amino acid modifications at any one or more of the following amino acid residues: Ser3, Leu17, Ala114, Trp111, Tyr117, Leu118, Pro156, Gly159, Gln160, Asn161, Pro162, Ser163, Ala164, Arg165, Ser166, Gln167, Lys168, Val169, Val170, Glu171, Ala172, Tyr179, Gln182, Lys187, His180, Asn181, Met209, Leu210, Arg211, Asn215, Lys284, Met285, Gln289, Val290, Ala309, Ser310, Lys22, Met23, Gly40, Asn80, Pro81, Lys82, Val112, Asn87, Asn88.

Preferably, X of the GDSX motif is L. Thus, preferably the parent enzyme comprises the amino acid motif GDSL.

Preferably the method of producing a variant lipid acyltransferase enzyme further comprises one or more of the following steps:

-   -   1) structural homology mapping or     -   2) sequence homology alignment.

Suitably, the structural homology mapping may comprise one or more of the following steps:

-   -   i) aligning a parent sequence with a structural model (1IVN.PDB)         shown in FIG. 52;     -   ii) selecting one or more amino acid residue within a 10 Å         sphere centred on the central carbon atom of the glycerol         molecule in the active site (see FIG. 53); and     -   iii) modifying one or more amino acids selected in accordance         with step (ii) in said parent sequence.

In one embodiment preferably the amino acid residue selected in within an 9, preferably within a 8, 7, 6, 5, 4, or 3 Å sphere centred on the central carbon atom of the glycerol molecule in the active site (see FIG. 53).

Suitably, the structural homology mapping may comprise one or more of the following steps:

-   -   i) aligning a parent sequence with a structural model (1IVN.PDB)         shown in FIG. 52;     -   ii) selecting one or more amino acids within a 10 Å sphere         centred on the central carbon atom of the glycerol molecule in         the active site (see FIG. 53);     -   iii) determining if one or more amino acid residues selected in         accordance with step (ii) are highly conserved (particularly are         active site residues and/or part of the GDSx motif and/or part         of the GANDY motif); and     -   iv) modifying one or more amino acids selected in accordance         with step (ii), excluding conserved regions identified in         accordance with step (iii) in said parent sequence.

In one embodiment preferably the amino acid residue selected in within an 9, preferably within a 8, 7, 6, 5, 4, or 3 Å sphere centred on the central carbon atom of the glycerol molecule in the active site (see FIG. 53).

Suitably, the sequence homology alignment may comprise one or more of the following steps:

-   -   i) selecting a first parent lipid acyltransferase;     -   ii) identifying a second related lipid acyltransferase having a         desirable activity;     -   iii) aligning said first parent lipid acyltransferase and the         second related lipid acyltransferase;     -   iv) identifying amino acid residues that differ between the two         sequences; and     -   v) modifying one or more of the amino acid residues identified         in accordance with step (iv) in said parent lipid         acyltransferase.

Suitably, the sequence homology alignment may comprise one or more of the following steps:

-   -   i) selecting a first parent lipid acyltransferase;     -   ii) identifying a second related lipid acyltransferase having a         desirable activity;     -   iii) aligning said first parent lipid acyltransferase and the         second related lipid acyltransferase;     -   iv) identifying amino acid residues that differ between the two         sequences;     -   v) determining if one or more amino acid residues selected in         accordance with step (iv) are highly conserved (particularly are         active site residues and/or part of the GDSx motif and/or part         of the GANDY motif); and     -   vi) modifying one or more of the amino acid residues identified         in accordance with step (iv) excluding conserved regions         identified in accordance with step (v) in said parent sequence.

Suitably, said first parent lipid acyltransferase may comprise any one of the following amino acid sequences: SEQ ID No. 2, SEQ ID No. 3, SEQ ID No. 4, SEQ ID No. 5, SEQ ID No. 6, SEQ ID No. 12, SEQ ID No. 14, SEQ ID No. 16, SEQ ID No. 18, SEQ ID No. 20, SEQ ID No. 22, SEQ ID No. 24, SEQ ID No. 26, SEQ ID No. 28, SEQ ID No. 30, SEQ ID No. 33, SEQ ID No. 34, SEQ ID No. 36, SEQ ID No. 37, SEQ ID No. 39, SEQ ID No. 41, SEQ ID No. 43 or SEQ ID No. 45.

Suitably, said second related lipid acyltransferase may comprise any one of the following amino acid sequences: SEQ ID No. 2, SEQ ID No. 3, SEQ ID No. 4, SEQ ID No. 5, SEQ ID No. 6, SEQ ID No. 12, SEQ ID No. 14, SEQ ID No. 16, SEQ ID No. 18, SEQ ID No. 20, SEQ ID No. 22, SEQ ID No. 24, SEQ ID No. 26, SEQ ID No. 28, SEQ ID No. 30, SEQ ID No. 33, SEQ ID No. 34, SEQ ID No. 36, SEQ ID No. 37, SEQ ID No. 39, SEQ ID No. 41, SEQ ID No. 43 or SEQ ID No. 45.

Suitably the variant enzyme may comprise at least one amino acid modification. In some embodiments, the variant enzyme may comprise at least 2, preferably at least 3, preferably at least 4, preferably at least 5, preferably at least 6, preferably at least 7, preferably at least 8, preferably at least 9, preferably at least 10 amino acid modifications compared with the parent enzyme.

In order to align a GDSx polypeptide sequence (parent sequence) with SEQ ID No. 2 (P01480), sequence alignment such as pairwise alignment can be used (http://www.ebi.ac.uk/emboss/align/index.html). Thereby, the equivalent amino acids in alternative parental GDSx polypeptides, which correspond to one or more of the following amino acids Ser3, Leu17, Ala114, Trp111, Tyr117, Leu118, Pro156, Gly159, Gln160, Asn161, Pro162, Ser163, Ala164, Arg165, Ser166, Gln167, Lys168, Val169, Val170, Glu171, Ala172, Tyr179, Gln182, Lys187, His180, Asn181, Met209, Leu210, Arg211, Asn215, Lys284, Met285, Gln289, Val290, Ala309, Ser310, Lys22, Met23, Gly40, Asn80, Pro81, Lys82, Val112, Asn87, Asn88 of SEQ ID No. 2 can be determined and modified. As the skilled person will readily appreciate, when using the emboss pairwise alignment, standard settings usually suffice. Corresponding residues can be identified using “needle” in order to make an alignment that covers the whole length of both sequences. However, it is also possible to find the best region of similarity between two sequences, using “water”.

Alternatively, particularly in instances where parent GDSx polypeptides share low homology with SEQ ID No. 2, the corresponding amino acids in alternative parental GDSx polypeptides which correspond to one or more of the following amino acids Ser3, Leu17, Ala114, Trp111, Tyr117, Leu118, Pro156, Gly159, Gln160, Asn161, Pro162, Ser163, Ala164, Arg165, Ser166, Gln167, Lys168, Val169, Val170, Glu171, Ala172, Tyr179, Gln182, Lys187, His180, Asn181, Met209, Leu210, Arg211, Asn215, Lys284, Met285, Gln289, Val290, Ala309, Ser310, Lys22, Met23, Gly40, Asn80, Pro81, Lys82, Val112, Asn87, Asn88 of SEQ ID No. 2 can be determined by structural alignment to the structural model of P10480, obtained by the structural alignment of P10480 crystal structure coordinates of 1IVN.PDB and 1DEO.PDB using the ‘Deep View Swiss-PDB viewer’ (obtained from www.expasy.org/spdbv/) (FIG. 53 and Example 1). Equivalent residues are identified as those overlapping or in closest proximity to the residues in the obtained structural model of P010480.

Alternatively, particularly in instances where a parent GDSx polypeptide shares a low homology with SEQ ID No. 2, the equivalent amino acids in alternative parental GDSx polypeptides, which correspond to one or more of the following amino acids Ser3, Leu17, Ala114, Trp111, Tyr117, Leu118, Pro156, Gly159, Gln160, Asn161, Pro162, Ser163, Ala164, Arg165, Ser166, Gln167, Lys168, Val169, Val170, Glu171, Ala172, Tyr179, Gln182, Lys187, His180, Asn181, Met209, Leu210, Arg211, Asn215, Lys284, Met285, Gln289, Val290, Ala309, Ser310, Lys22, Met23, Gly40, Asn80, Pro81, Lys82, Val112, Asn87, Asn88 of SEQ ID No. 2 can be determined from an alignment obtained from the PFAM database (PFAM consensus) modified based on the structural alignment as shown in Alignment 1 (FIG. 55). The modification based on the structural models may be necessary to slightly shift the alignment in order to ensure a best fit overlap. Alignment 1 (FIG. 55) provides guidance in this regard.

Suitably the variant enzyme may be prepared using site directed mutagenesis.

Alternatively, one can introduce mutations randomly for instance using a commercial kit such as the GeneMorph PCR mutagenesis kit from Stratagene, or the Diversify PCR random mutagenesis kit from Clontech. EP 0 583 265 refers to methods of optimising PCR based mutagenesis, which can also be combined with the use of mutagenic DNA analogues such as those described in EP 0 866 796. Error prone PCR technologies are suitable for the production of variants of lipid acyl transferases with preferred characteristics. WO0206457 refers to molecular evolution of lipases.

A third method to obtain novel sequences is to fragment non-identical nucleotide sequences, either by using any number of restriction enzymes or an enzyme such as Dnase I, and reassembling full nucleotide sequences coding for functional proteins (hereinafter referred to as “shuffling”). Alternatively one can use one or multiple non-identical nucleotide sequences and introduce mutations during the reassembly of the full nucleotide sequence. DNA shuffling and family shuffling technologies are suitable for the production of variants of lipid acyl transferases with preferred characteristics. Suitable methods for performing ‘shuffling’ can be found in EP0 752 008, EP1 138 763, EP1 103 606. Shuffling can also be combined with other forms of DNA mutagenesis as described in U.S. Pat. No. 6,180,406 and WO 01/34835.

Thus, it is possible to produce numerous site directed or random mutations into a nucleotide sequence, either in vivo or in vitro, and to subsequently screen for improved functionality of the encoded variant polypeptide by various means.

As a non-limiting example, In addition, mutations or natural variants of a polynucleotide sequence can be recombined with either the wild type or other mutations or natural variants to produce new variants. Such new variants can also be screened for improved functionality of the encoded polypeptide.

Suitably, the variant lipid acyltransferase according to the present invention retains at least 70%, preferably at least 80%, preferably at least 90%, preferably at least 95%, preferably at least 97%, preferably at least 99% homology with the parent enzyme.

Suitable parent enzymes may include any enzyme with esterase or lipase activity.

Preferably, the parent enzyme aligns to the pfam00657 consensus sequence.

In a preferable embodiment a variant lipid acyltransferase enzyme retains or incorporates at least one or more of the pfam00657 consensus sequence amino acid residues found in the GDSx, GANDY and HPT blocks.

Enzymes, such as lipases with no or low lipid acyltransferase activity in an aqueous environment may be mutated using molecular evolution tools to introduce or enhance the transferase activity, thereby producing a variant lipid acyltransferase enzyme with significant transferase activity suitable for use in the compositions and methods of the present invention.

Suitably, the lipid acyltransferase for use in the invention may be a variant with enhanced enzyme activity on polar lipids, preferably glycolipids, when compared to the parent enzyme. Preferably, such variants also have low or no activity on lyso polar lipids. The enhanced activity on polar lipids, preferably glycolipids may be the result of hydrolysis and/or transferase activity or a combination of both.

Variant lipid acyltransferases for use in the invention may have decreased activity on triglycerides, and/or monoglycerides and/or diglycerides compared with the parent enzyme.

Suitably the variant enzyme may have no activity on triglycerides and/or monoglycerides and/or diglycerides.

When referring to specific amino acid residues herein the numbering is that obtained from alignment of the variant sequence with the reference sequence shown as SEQ ID No. 2.

In one aspect preferably the variant enzyme comprises one or more of the following amino acid substitutions:

-   -   S3A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y;         and/or     -   L17A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or         Y; and/or     -   S18A, C, D, E, F, H, I, K, L, M, N, P, Q, R, T, W, or Y; and/or     -   K22A, C, D, E, F, G, H, I, L, M, N, P, Q, R, S, T, V, W, or Y;         and/or     -   M23A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or         Y; and/or     -   G40A, C, D, E, F, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y;         and/or     -   N80A, C, D, E, F, G, H, I, K, L, M, P, Q, R, S, T, V, W, or Y;         and/or     -   N87A, C, D, E, F, G, H, I, K, L, M, P, Q, R, S, T, V, W, or Y;         and/or     -   N88A, C, D, E, F, G, H, I, K, L, M, P, Q, R, S, T, V, W, or Y;         and/or     -   P81A, C, D, E, F, G, H, I, K, L, M, N, Q, R, S, T, V, W, or Y;         and/or     -   L82A, C, D, E, F, G, H, I, L, M, N, P, Q, R, S, T, V, W, or Y;         and/or     -   V112A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, W, or Y;         and/or     -   A114C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y;         and/or     -   W111A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, or Y;         and/or     -   Y117A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, or W;         and/or     -   L118A, C, D, E, F, G, H, I, K, M, N, P, Q, R, S, T, V, W, or Y;         and/or     -   P156A, C, D, E, F, G, H, I, K, L, M, N, Q, R, S, T, V, W, or Y;         and/or     -   G159A, C, D, E, F, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y;         and/or     -   Q160A, C, D, E, F, G, H, I, K, L, M, N, P, R, S, T, V, W, or Y;         and/or     -   N161A, C, D, E, F, G, H, I, K, L, M P, Q, R, S, T, V, W, or Y;         and/or     -   P162A, C, D, E, F, G, H, I, K, L, M, N, Q, R, S, T, V, W, or Y;         and/or     -   S163A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, T, V, W, or Y;         and/or     -   A164C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y;         and/or     -   R165A, C, D, E, F, G, H, I, K, L, M, N, P, Q, S, T, V, W, or Y;         and/or     -   S166A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, T, V, W, or Y;         and/or     -   Q167A, C, D, E, F, G, H, I, K, L, M, N, P, R, S, T, V, W, or Y;         and/or     -   K168A, C, D, E, F, G, H, I, L, M, N, P, Q, R, S, T, V, W, or Y;         and/or     -   V169A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, W, or Y;         and/or     -   V170A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, W, or Y;         and/or     -   E171A, C, D, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y;         and/or     -   A172C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y;         and/or     -   Y179A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, or W;         and/or     -   H180A, C, D, E, F, G, I, K, L, M, P, Q, R, S, T, V, W, or Y;         and/or     -   N181A, C, D, E, F, G, H, I, K, L, M, P, Q, R, S, T, V, W, or Y;         and/or     -   Q182A, C, D, E, F, G, H, I, K, L, M, N, P, R, S, T, V, W, or Y;         and/or     -   M209A, C, D, E, F, G, H, I, K, L, N, P, Q, R, S, T, V, W, or Y;         and/or     -   L210A, C, D, E, F, G, H, I, K, M, N, P, Q, R, S, T, V, W, or Y;         and/or     -   R211A, C, D, E, F, G, H, I, K, L, M, N, P, Q, S, T, V, W, or Y;         and/or     -   N215A, C, D, E, F, G, H, I, K, L, M, P, Q, R, S, T, V, W, or Y;         and/or     -   Y230A, C, D, E, G, H, I, K, L, M, N, P, Q, R, S, T, V or W;         and/or     -   K284A, C, D, E, F, G, H, I, L, M, N, P, Q, R, S, T, V, W, or Y;         and/or     -   M285A, C, D, E, F, G, H, I, K, L, N, P, Q, R, S, T, V, W, or Y;         and/or     -   Q289A, C, D, E, F, G, H, I, K, L, M, N, P, R, S, T, V, W, or Y;         and/or     -   V290A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, W, or Y;         and/or     -   A309C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y;         and/or     -   S310A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, T, V, W, or Y;         and/or

In addition or alternatively thereto there may be one or more C-terminal extensions. Preferably the additional C-terminal extension is comprised of one or more aliphatic amino acids, preferably a non-polar amino acid, more preferably of I, L, V or G. Thus, the present invention further provides for a variant enzyme comprising one or more of the following C-terminal extensions: 318I, 318L, 318V, 318G.

When it is the case that the residues in the parent backbone differ from those in P10480 (SEQ ID No. 2), as determined by homology alignment and/or structural alignment to P10480 and/or 1IVN, it may be desirable to replace the residues which align to any one or more of the following amino acid residues in P10480 (SEQ ID No. 2): Ser3, Leu17, Ala114, Trp111, Tyr117, Leu118, Pro156, Gly159, Gln160, Asn161, Pro162, Ser163, Ala164, Arg165, Ser166, Gln167, Lys168, Val169, Val170, Glu171, Ala172, Tyr179, Gln182, Lys187, His180, Asn181, Met209, Leu210, Arg211, Asn215, Lys284, Met285, Gln289, Val290, Ala309, Ser310, Lys22, Met23, Gly40, Asn80, Pro81, Lys82, Val112, Ser18, Asn87, Asn88, −318, Tyr230, Tyr30, with the residue found in P10480.

Preferably, the His amino acid at residue 180 is substituted for one of the following A, D, E, F, G, I, K, L, P, R, V, W, or Y.

Preferably, the Gln amino acid at residue 182 is substituted for a polar amino acid, most preferably K, R, D, or E.

Preferably, the Tyr amino acid at residue 230 is substituted for one of the following amino acids A, C, D, E, G, I, K, L, M, N, P, Q, R, S, T, V, or Y

In one aspect preferably the variant enzyme comprises one or more of the following amino acid substitutions: S3T, Q182K, E309A, S310E.

In a further aspect, preferably the variant enzyme comprises a C-terminal addition, namely −318G.

Suitably, the variant enzyme may comprise one or more of the following modifications: S3T, Q182K, E309A, S310E, −318G.

Variant enzymes which have an increased hydrolytic activity against a polar lipid may also have an increased transferase activity from a polar lipid.

Variant enzymes which have an increased hydrolytic activity against a phospholipid, such as phosphatidylcholine (PC) may also have an increased transferase activity from a phospholipid.

Variant enzymes which have an increased hydrolytic activity against a galactolipid, such as DGDG, may also have an increased transferase activity from a galactolipid.

Variants enzymes which have an increased transferase activity from a phospholipid, such as phosphatidylcholine (PC), may also have an increased hydrolytic activity against a phospholipid.

Variants enzymes which have an increased transferase activity from a galactolipid, such as DGDG, may also have an increased hydrolytic activity against a galactolipid.

Variants enzymes which have an increased transferase activity from a polar lipid may also have an increased hydrolytic activity against a polar lipid.

Suitably, one or more of the following sites may be involved in substrate binding: Leu17; Ala114; Tyr179; His180; Asn181; Met209, Leu210; Arg211; Asn215; Lys284; Met285; Gln289; Val290.

The variant enzyme in accordance with the present invention may have one or more of the following functionalities compared with the parent enzyme:

-   -   i) improved activity towards a phospholipid, such as         phosphatidylcholine;     -   ii) improved activity towards a galactolipid, such as DGDG;     -   iii) improved specificity towards a galactolipid, in particular         DGDG;     -   iv) improved galactolipid:phospholipid ratio);     -   v) improved transferase activity with a phospholipid, such as         phosphatidylcholine, as the lipid acyl donor;     -   vi) improved transferase activity with a galactolipid, such as         DGDG, as the lipid acyl donor

The following modifications may result in variants having an improved activity towards a polar lipid substrate (phospholipids and/or galactolipids):

-   -   S3A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y,         preferably S3 is substituted with an aliphatic amino acid or one         of the following residues S3T, S3N, S3Q, S3K, S3R, S3P, S3M;         and/or     -   D157A, C, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y,         preferably D157 is substituted with a polar uncharged amino         acid, preferably with C, S, T or M, more preferably C; and/or     -   Q182A, C, D, E, F, G, H, I, K, L, M, N, Q, P, R, S, T, V, W, or         Y, preferably Q182 is substituted with an aliphatic amino acid         residue, preferably a polar amino acid, more preferably a polar         charged amino acid, more preferably D or E, most preferably D;         and/or     -   A309A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or         Y, preferably A309 is substituted with an aliphatic residue,         preferably a non-polar residue, preferably G, A, or P, more         preferably A.

The following modifications may result in variants having an improved activity towards a galactolipid, such as DGDG:

-   -   S3A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y,         preferably S3 is substituted with an aliphatic amino acid or one         of the following amino acid residues S3G, S3A, S3T, S3N, S3Q,         S3K, S3R, S3P, S3M, or a polar charged amino acid, preferably C,         S, T, M, N or Q, more preferably N or Q; and/or     -   Y230A, C, D, E, G, H, I, K, L, M, N, P, Q, R, S, T, V, or W,         preferably Y230 is substituted with an aliphatic amino acid or         one of the following amino acid residues G, D, T, V, R or M,         more preferably G, D, T, V, R or M, more preferably G or T;         and/or     -   Q182A, C, D, E, F, G, H, I, K, L, M, N, Q, P, R, S, T, V, W, or         Y, preferably Q182 is substituted with an aliphatic amino acid,         preferably a polar amino acid, preferably a polar charged amino         acid, more preferably D or E, most preferably D; and/or     -   A309A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or         Y, preferably A309 is substituted with an aliphatic amino acid,         preferably a non-polar amino acid, preferably G, A, or P, more         preferably A; and/or     -   A C-terminal addition (−318) of at least one amino acid,         preferably one amino acid, wherein the additional amino acid is         preferably an aliphatic amino acid, preferably a non-polar amino         acid, more preferably I, L or V. I

The following modifications may result in variants having an improved specificity towards a galactolipid, in particular DGDG:

-   -   Y230A, C, D, E, G, H, I, K, L, M, N, P, Q, R, S, T, V, or W,         preferably Y230 is substituted with an aliphatic amino acid or         one of the following amino acid residues G, D, T, V, R or M,         more preferably G, D, T, V, R or M;

The following modifications may result in variants having an improved galactolipid:phospholipid ratio:

-   -   Y230A, C, D, E, G, H, I, K, L, M, N, P, Q, R, S, T, V, or W,         preferably Y230 is substituted with an aliphatic amino acid or         one of the following amino acid residues G, D, T, V, R or M,         more preferably G, D, T, V, R or M.

The following modifications may result in variants having an improved activity with a phospholipid, such as phosphatidylcholine, as the lipid acyl donor:

-   -   A309A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or         Y, preferably A309 is substituted with an aliphatic amino acid,         preferably a non-polar amino acid, preferably G, A, or P, more         preferably A; and/or     -   S3A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y,         preferably SA is substituted with a polar uncharged and/or polar         charged amino acid, preferably one of the following amino acids         residues S3T, S3N, S3Q, S3K, S3R, S3P, S3M, more preferably S3Q,         S3K, or S3R.

The following modifications may result in variants having an improved transferase activity with a phospholipid, such as phosphatidylcholine, as the lipid acyl donor:

-   -   S3A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y,         preferably SA is substituted with a polar uncharged and/or polar         charged amino acid pore preferably one of the following amino         acids residues S3T, S3N, S3Q, S3D, S3K, S3R, S3P, S3M; and/or     -   Q182A, C, D, E, F, G, H, I, K, L, M, N, Q, P, R, S, T, V, W, or         Y, preferably Q182 is substituted with an aliphatic amino acid         residue, preferably a polar amino acid, preferably a polar         charged amino acid, more preferably D or E, most preferably D;         and/or     -   A309A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or         Y, preferably A309 is substituted with an aliphatic residue,         preferably a non-polar residue, preferable G, A, or P, more         preferably A.

The following modifications may result in variants having an improved transferase activity using a galactolipid acyl, such as DGDG, as the lipid acyl donor:

-   -   Q182A, C, D, E, F, G, H, I, K, L, M, N, Q, P, R, S, T, V, W, or         Y, preferably Q182 is substituted by an aliphatic amino acid         residue, preferably a polar amino acid, preferably a polar         charged amino acid, more preferably D or E, most preferably D;         and/or     -   Y230A, C, D, E, G, H, I, K, L, M, N, P, Q, R, S, T, V, or W,         preferably Y230 is substituted with an aliphatic amino acid or         one of the following amino acid residues G, D, T, V, R or M,         more preferably G, D, T, V, R or M, more preferably G or T;         and/or     -   A309A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or         Y, preferably A309 is substituted with an aliphatic residue,         preferably a non-polar residue, preferable G, A, or P, more         preferably A.

The following modifications may result in variants having an improved transferase activity with a polar lipid, such as a galactolipid (e.g. DGDG) and/or a phospholipid (e.g. phosphatidylcholine) as the lipid acyl donor:

-   -   S3A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y,         preferably S3 is substituted with a polar uncharged and/or polar         charged amino acid, more preferably one of the following amino         acids residues S3T, S3N, S3Q, S3D, S3K, S3R, S3P, S3M; and/or     -   Y230A, C, D, E, G, H, I, K, L, M, N, P, Q, R, S, T, V, or W,         preferably Y230 is substituted with an aliphatic amino acid or         one of the following amino acid residues G, D, T, V, R or M,         more preferably G, D, T, V, R or M, more preferably G or T;         and/or     -   Q182A, C, D, E, F, G, H, I, K, L, M, N, Q, P, R, S, T, V, W, or         Y, preferably an aliphatic amino acid residue, preferably a         polar amino acid, preferably a polar charged amino acid, more         preferably D or E, most preferably D; and/or     -   S3A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y,         preferably S3 is substituted with a polar uncharged and/or polar         charged amino acid, more preferably one of the following amino         acids residues S3T, S3N, S3Q, S3D, S3K, S3R, S3P, S3M; and/or     -   A309A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or         Y, preferably an aliphatic residue, preferably a non-polar         residue, preferable G, A, or P, more preferably A.

The following modifications result in variants having improved activity towards PC:

-   -   S3N, Q, K, R, P, and/or M

The following modifications result in variants having improved activity towards DGDG:

-   -   K187D, E309A, Y230T, Y230G, S3Q

The following modifications result in variants having improved specificity towards DGDG:

-   -   K187D, K187D, Y230G, Y230T, Y230R, Y230M, Y230V, D157C, E309A,         G218I

The following modifications result in variants having improved transferase activity with PC as the acyl donor:

-   -   S3K, S3R, S3Q, S3N, S3P, S3M

The following modifications result in variants having improved transferase activity with DGDG as the acyl donor:

-   -   Y230T, K187D, Y230G, E309A

As noted above, when referring to specific amino acid residues herein the numbering is that obtained from alignment of the variant sequence with the reference sequence shown as SEQ ID No. 2.

For the avoidance of doubt, when a particular amino acid is taught at a specific site, for instance K187 for instance, this refers to the specific amino acid at residue number 187 in SEQ ID No. 2. However, the amino acid residue at site 187 in a different parent enzyme may be different from lysine.

Thus, when taught to substitute an amino acid at residue 187, although reference may be made to K187 it would be readily understood by the skilled person that when the parent enzyme is other than that shown in SEQ ID No. 2, the amino acid being substituted may not be lysine. It is, therefore, possible that when substituting an amino acid sequence in a parent enzyme which is not the enzyme having the amino acid sequence shown as SEQ ID No. 2, the new (substituting) amino acid may be the same as that taught in SEQ ID No. 2. This may be the case, for instance, where the amino acid at say residue 187 is not lysine and is, therefore different from the amino acid at residue 187 in SEQ ID No. 2. In other words, at residue 187 for example, if the parent enzyme has at that position an amino acid other than lysine, this amino acid may be substituted with lysine in accordance with the present invention.

The term “lipid acyltransferase” as used herein means an enzyme which has acyltransferase activity (generally classified as E.C. 2.3.1.x in accordance with the Enzyme Nomenclature Recommendations (1992) of the Nomenclature Committee of the International Union of Biochemistry and Molecular Biology), whereby the enzyme is capable of transferring an acyl group from a lipid to one or more acceptor substrates, such as one or more of the following: a sterol; a stanol; a carbohydrate; a protein; a protein subunit; glycerol.

Preferably, the lipid acyltransferase variant according to the present invention and/or for use in the methods and/or uses of the present invention is capable of transferring an acyl group from a lipid (as defined herein) to one or more of the following acyl acceptor substrates: a sterol, a stanol, a carbohydrate, a protein or subunits thereof, or a glycerol.

For some aspects the “acyl acceptor” according to the present invention may be any compound comprising a hydroxy group (—OH), such as for example, polyvalent alcohols, including glycerol; sterol; stanols; carbohydrates; hydroxy acids including fruit acids, citric acid, tartaric acid, lactic acid and ascorbic acid; proteins or a sub-unit thereof, such as amino acids, protein hydrolysates and peptides (partly hydrolysed protein) for example; and mixtures and derivatives thereof. Preferably, the “acyl acceptor” according to the present invention is not water.

In one embodiment, the acyl acceptor is preferably not a monoglyceride and/or a diglyceride.

In one aspect, preferably the variant enzyme is capable of transferring an acyl group from a lipid to a sterol and/or a stanol.

In one aspect, preferably the variant enzyme is capable of transferring an acyl group from a lipid to a carbohydrate.

In one aspect, preferably the variant enzyme is capable of transferring an acyl group from a lipid to a protein or a subunit thereof. Suitably the protein subunit may be one or more of the following: an amino acid, a protein hydrolysate, a peptide, a dipeptide, an oligopeptide, a polypeptide.

Suitably in the protein or protein subunit the acyl acceptor may be one or more of the following constituents of the protein or protein subunit: a serine, a threonine, a tyrosine, or a cysteine.

When the protein subunit is an amino acid, suitably the amino acid may be any suitable amino acid. Suitably the amino acid may be one or more of a serine, a threonine, a tyrosine, or a cysteine for example.

In one aspect, preferably the variant enzyme is capable of transferring an acyl group from a lipid to glycerol.

In one aspect, preferably the variant enzyme is capable of transferring an acyl group from a lipid to a hydroxy acid.

In one aspect, preferably the variant enzyme is capable of transferring an acyl group from a lipid to a polyvalent alcohol.

In one aspect, the variant lipid acyltransferase may, as well as being able to transfer an acyl group from a lipid to a sterol and/or a stanol, additionally be able to transfer the acyl group from a lipid to one or more of the following: a carbohydrate, a protein, a protein subunit, glycerol.

Preferably, the lipid substrate upon which the variant lipid acyltransferase according to the present invention acts is one or more of the following lipids: a phospholipid, such as a lecithin, e.g. phosphatidylcholine, a triacylglyceride, a cardiolipin, a diglyceride, or a glycolipid, such as digalactosyldiglyceride (DGDG) for example. This lipid substrate may be referred to herein as the “lipid acyl donor”. The term lecithin as used herein encompasses phosphatidylcholine, phosphatidylethanolamine, phosphatidylinositol, phosphatidylserine and phosphatidylglycerol.

For some aspects, preferably the lipid substrate upon which the variant lipid acyltransferase acts is a phospholipid, such as lecithin, for example phosphatidylcholine.

For some aspects, preferably the lipid substrate is a glycolipid, such as DGDG for example.

Preferably the lipid substrate is a food lipid, that is to say a lipid component of a foodstuff.

For some aspects, preferably the variant lipid acyltransferase according to the present invention is incapable, or substantially incapable, of acting on a triglyceride and/or a 1-monoglyceride and/or 2-monoglyceride.

Suitably, the lipid substrate or lipid acyl donor may be one or more lipids present in one or more of the following substrates: fats, including lard, tallow and butter fat; oils including oils extracted from or derived from palm oil, sunflower oil, soya bean oil, safflower oil, cotton seed oil, ground nut oil, corn oil, olive oil, peanut oil, coconut oil, and rape seed oil. Lecithin from soya, rape seed or egg yolk is also a suitable lipid substrate. The lipid substrate may be an oat lipid or other plant based material containing galactolipids.

In one aspect the lipid acyl donor is preferably lecithin (such as phosphatidylcholine) in egg yolk.

For some aspects of the present invention, the lipid may be selected from lipids having a fatty acid chain length of from 8 to 22 carbons.

For some aspects of the present invention, the lipid may be selected from lipids having a fatty acid chain length of from 16 to 22 carbons, more preferably of from 16 to 20 carbons.

For some aspects of the present invention, the lipid may be selected from lipids having a fatty acid chain length of no greater than 14 carbons, suitably from lipids having a fatty acid chain length of from 4 to 14 carbons, suitably 4 to 10 carbons, suitably 4 to 8 carbons.

Suitably, the variant lipid acyltransferase according to the present invention may exhibit one or more of the following lipase activities: glycolipase activity (E.C. 3.1.1.26), triacylglycerol lipase activity (E.C. 3.1.1.3), phospholipase A2 activity (E.C. 3.1.1.4) or phospholipase A1 activity (E.C. 3.1.1.32). The term “glycolipase activity” as used herein encompasses “galactolipase activity”.

Suitably, the variant lipid acyltransferase according to the present invention may have at least one or more of the following activities: glycolipase activity (E.C. 3.1.1.26) and/or phospholipase A1 activity (E.C. 3.1.1.32) and/or phospholipase A2 activity (E.C. 3.1.1.4).

For some aspects, the variant lipid acyltransferase according to the present invention may have at least glycolipase activity (E.C. 3.1.1.26).

Suitably, for some aspects the variant lipid acyltransferase according to the present invention may be capable of transferring an acyl group from a glycolipid and/or a phospholipid to one or more of the following acceptor substrates: a sterol, a stanol, a carbohydrate, a protein, glycerol.

For some aspects, preferably the variant lipid acyltransferase according to the present invention is capable of transferring an acyl group from a glycolipid and/or a phospholipid to a sterol and/or a stanol to form at least a sterol ester and/or a stanol ester.

For some aspects, preferably the variant lipid acyltransferase according to the present invention is capable of transferring an acyl group from a glycolipid and/or a phospholipid to a carbohydrate to form at least a carbohydrate ester.

For some aspects, preferably the variant lipid acyltransferase according to the present invention is capable of transferring an acyl group from a glycolipid and/or a phospholipid to a protein to form at least protein ester (or a protein fatty acid condensate).

For some aspects, preferably the variant lipid acyltransferase according to the present invention is capable of transferring an acyl group from a glycolipid and/or a phospholipid to glycerol to form at least a diglyceride and/or a monoglyceride.

For some aspects, preferably the variant lipid acyltransferase according to the present invention does not exhibit triacylglycerol lipase activity (E.C. 3.1.1.3).

In some aspects, the variant lipid acyltransferase may be capable of transferring an acyl group from a lipid to a sterol and/or a stanol. Thus, in one embodiment the “acyl acceptor” according to the present invention may be either a sterol or a stanol or a combination of both a sterol and a stanol.

In one embodiment suitably the sterol and/or stanol may comprise one or more of the following structural features:

-   -   i) a 3-beta hydroxy group or a 3-alpha hydroxy group; and/or     -   ii) A:B rings in the cis position or A:B rings in the trans         position or C₅-C₆ is unsaturated.

Suitable sterol acyl acceptors include cholesterol and phytosterols, for example alpha-sitosterol, beta-sitosterol, stigmasterol, ergosterol, campesterol, 5,6-dihydrosterol, brassicasterol, alpha-spinasterol, beta-spinasterol, gamma-spinasterol, deltaspinasterol, fucosterol, dimosterol, ascosterol, serebisterol, episterol, anasterol, hyposterol, chondrillasterol, desmosterol, chalinosterol, poriferasterol, clionasterol, sterol glycosides, and other natural or synthetic isomeric forms and derivatives.

In one aspect of the present invention suitably more than one sterol and/or stanol may act as the acyl acceptor, suitably more than two sterols and/or stanols may act as the acyl acceptor. In other words, in one aspect of the present invention, suitably more than one sterol ester and/or stanol ester may be produced. Suitably, when cholesterol is the acyl acceptor one or more further sterols or one or more stanols may also act as the acyl acceptor. Thus, in one aspect, the present invention provides a method for the in situ production of both a cholesterol ester and at least one sterol or stanol ester in combination. In other words, the lipid acyltransferase for some aspects of the present invention may transfer an acyl group from a lipid to both cholesterol and at least one further sterol and/or at least one stanol.

In one aspect, preferably the sterol acyl acceptor is one or more of the following: alpha-sitosterol, beta-sitosterol, stigmasterol, ergosterol and campesterol.

In one aspect, preferably the sterol acyl acceptor is cholesterol. When it is the case that cholesterol is the acyl acceptor for the variant lipid acyltransferase, the amount of free cholesterol in the foodstuff is reduced as compared with the foodstuff prior to exposure to the variant lipid acyltransferase and/or as compared with an equivalent foodstuff which has not been treated with the variant lipid acyltransferase.

Suitable stanol acyl acceptors include phytostanols, for example beta-sitostanol or ss-sitostanol.

In one aspect, preferably the sterol and/or stanol acyl acceptor is a sterol and/or a stanol other than cholesterol.

In some aspects, the foodstuff prepared in accordance with the present invention may be used to reduce blood serum cholesterol and/or to reduce low density lipoprotein. Blood serum cholesterol and low density lipoproteins have both been associated with certain diseases in humans, such as atherosclerosis and/or heart disease for example. Thus, it is envisaged that the foodstuffs prepared in accordance with the present invention may be used to reduce the risk of such diseases.

Thus, in one aspect the present invention provides the use of a foodstuff according to the present invention for use in the treatment and/or prevention of atherosclerosis and/or heart disease.

In a further aspect, the present invention provides a medicament comprising a foodstuff according to the present invention.

In a further aspect, the present invention provides a method of treating and/or preventing a disease in a human or animal patient which method comprising administering to the patient an effective amount of a foodstuff according to the present invention.

Suitably, the sterol and/or the stanol “acyl acceptor” may be found naturally within the foodstuff. Alternatively, the sterol and/or the stanol may be added to the foodstuff. When it is the case that a sterol and/or a stanol is added to the foodstuff, the sterol and/or stanol may be added before, simultaneously with, and/or after the addition of the lipid acyltransferase according to the present invention. Suitably, the present invention may encompass the addition of exogenous sterols/stanols, particularly phytosterols/phytostanols, to the foodstuff prior to or simultaneously with the addition of the variant enzyme according to the present invention.

For some aspects, one or more sterols present in the foodstuff may be converted to one or more stanols prior to or at the same time as the variant lipid acyltransferase is added according to the present invention. Any suitable method for converting sterols to stanols may be employed. For example, the conversion may be carried out by chemical hydrogenation for example. The conversion may be conducted prior to the addition of the variant lipid acyltransferase in accordance with the present invention or simultaneously with the addition of the variant lipid acyltransferase in accordance with the present invention. Suitably enzymes for the conversion of sterol to stanols are taught in WO00/061771.

Suitably the present invention may be employed to produce phytostanol esters in situ in a foodstuff. Phytostanol esters have increased solubility through lipid membranes, bioavailability and enhanced health benefits (see for example WO92/99640).

In some embodiments of the present invention the stanol ester and/or the sterol ester may be a flavouring and/or a texturiser. In which instances, the present invention encompasses the in situ production of flavourings and/or texturisers.

For some aspects of the present invention, the variant lipid acyltransferase according to the present invention may utilise a carbohydrate as the acyl acceptor. The carbohydrate acyl acceptor may be one or more of the following: a monosaccharide, a disaccharide, an oligosaccharide or a polysaccharide. Preferably, the carbohydrate is one or more of the following: glucose, fructose, anhydrofructose, maltose, lactose, sucrose, galactose, xylose, xylooligosacharides, arabinose, maltooligosaccharides, tagatose, microthecin, ascopyrone P, ascopyrone T, cortalcerone.

Suitably, the carbohydrate “acyl acceptor” may be found naturally within the foodstuff. Alternatively, the carbohydrate may be added to the foodstuff. When it is the case that the carbohydrate is added to the foodstuff, the carbohydrate may be added before, simultaneously with, and/or after the addition of the variant lipid acyltransferase according to the present invention.

Carbohydrate esters can function as valuable emulsifiers in foodstuffs. Thus, when it is the case that the enzyme functions to transfer the acyl group to a sugar, the invention encompasses the production of a second in situ emulsifier in the foodstuff.

In some embodiments, the variant lipid acyltransferase may utilise both a sterol and/or stanol and a carbohydrate as an acyl acceptor.

The utilisation of a variant lipid acyltransferase which can transfer the acyl group to a carbohydrate as well as to a sterol and/or a stanol is particularly advantageous for foodstuffs comprising eggs. In particular, the presence of sugars, in particular glucose, in eggs and egg products is often seen as disadvantageous. Egg yolk may comprise up to 1% glucose. Typically, egg or egg based products may be treated with glucose oxidase to remove some or all of this glucose. However, in accordance with the present invention this unwanted sugar can be readily removed by “esterifying” the sugar to form a sugar ester.

For some aspects of the present invention, the variant lipid acyltransferase according to the present invention may utilise a protein as the acyl acceptor. Suitably, the protein may be one or more of the proteins found in a food product, for example in a dairy product and/or a meat product. By way of example only, suitable proteins may be those found in curd or whey, such as lactoglobulin. Other suitable proteins include ovalbumin from egg, gliadin, glutenin, puroindoline, lipid transfer proteins from grains, and myosin from meat.

Preferably, the parent lipid acyltransferase enzyme according to the present invention may be characterised using the following criteria:

-   -   (i) the enzyme possesses acyl transferase activity which may be         defined as ester transfer activity whereby the acyl part of an         original ester bond of a lipid acyl donor is transferred to an         acyl acceptor to form a new ester; and     -   (ii) the enzyme comprises the amino acid sequence motif GDSX,         wherein X is one or more of the following amino acid residues L,         A, V, I, F, Y, H, Q, T, N, M or S.

Preferably, X of the GDSX motif is L. Thus, preferably the enzyme according to the present invention comprises the amino acid sequence motif GSDL.

The GDSX motif is comprised of four conserved amino acids. Preferably, the serine within the motif is a catalytic serine of the lipid acyltransferase enzyme. Suitably, the serine of the GDSX motif may be in a position corresponding to Ser-16 in Aeromonas hydrophila lipolytic enzyme taught in Brumlik & Buckley (Journal of Bacteriology April 1996, Vol. 178, No. 7, p 2060-2064).

To determine if a protein has the GDSX motif according to the present invention, the sequence is preferably compared with the hidden markov model profiles (HMM profiles) of the pfam database.

Pfam is a database of protein domain families. Pfam contains curated multiple sequence alignments for each family as well as profile hidden Markov models (profile HMMs) for identifying these domains in new sequences. An introduction to Pfam can be found in Bateman A et al. (2002) Nucleic Acids Res. 30; 276-280. Hidden Markov models are used in a number of databases that aim at classifying proteins, for review see Bateman A and Haft D H (2002) Brief Bioinform 3; 236-245.

http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&list uids=122 30032&dopt=Abstract

http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&list uids=117 52314&dopt=Abstract

For a detailed explanation of hidden Markov models and how they are applied in the Pfam database see Durbin R, Eddy S, and Krogh A (1998) Biological sequence analysis; probabilistic models of proteins and nucleic acids. Cambridge University Press, ISBN 0-521-62041-4. The Hammer software package can be obtained from Washington University, St Louis, USA.

Alternatively, the GDSX motif can be identified using the Hammer software package, the instructions are provided in Durbin R, Eddy S, and Krogh A (1998) Biological sequence analysis; probabilistic models of proteins and nucleic acids. Cambridge University Press, ISBN 0-521-62041-4 and the references therein, and the HMMER2 profile provided within this specification.

The PFAM database can be accessed, for example, through several servers which are currently located at the following websites.

-   -   http://www.sanger.ac.uk/Software/Pfam/index.shtml     -   http://pfam.wustl.edu/     -   http://pfam.jouy.inra.fr/     -   http://pfam.cgb.ki.se/

The database offers a search facility where one can enter a protein sequence. Using the default parameters of the database the protein sequence will then be analysed for the presence of Pfam domains. The GDSX domain is an established domain in the database and as such its presence in any query sequence will be recognised. The database will return the alignment of the Pfam00657 consensus sequence to the query sequence.

A multiple alignment, including Aeromonas salmonicida or Aeromonas hydrophila can be obtained by:

-   -   a) manual         -   obtain an alignment of the protein of interest with the             Pfam00657 consensus sequence and obtain an alignment of             P10480 with the Pfam00657 consensus sequence following the             procedure described above;     -   or     -   b) through the database         -   After identification of the Pfam00657 consensus sequence the             database offers the option to show an alignment of the query             sequence to the seed alignment of the Pfam00657 consensus             sequence. P10480 is part of this seed alignment and is             indicated by GCAT_AERHY. Both the query sequence and P10480             will be displayed in the same window.

The Aeromonas hydrophila reference sequence:

The residues of Aeromonas hydrophila GDSX lipase are numbered in the NCBI file P10480, the numbers in this text refer to the numbers given in that file which in the present invention is used to determine specific amino acids residues which, in a preferred embodiment are present in the lipid acyltransferase enzymes of the invention.

The Pfam alignment was performed (FIG. 33 and FIG. 34):

The following conserved residues can be recognised and in a preferable embodiment may be present in the variant enzymes for use in the compositions and methods of the invention; Block 1 - GDSX block hid hid hid hid Gly Asp Ser hid  28  29  30  31  32  33  34  35 Block 2 - GANDY block hid Gly hid Asn Asp hid 130 131 132 133 134 135 Block 3 - HPT block His 309

Where ‘hid’ means a hydrophobic residue selected from Met, Ile, Leu, Val, Ala, Gly, Cys, His, Lys, Trp, Tyr, Phe.

Preferably the parent and/or variant lipid acyltransferase enzyme for use in the compositions/methods of the invention can be aligned using the Pfam00657 consensus sequence.

Preferably, a positive match with the hidden markov model profile (HMM profile) of the pfam00657 domain family indicates the presence of the GDSL or GDSX domain according to the present invention.

Preferably when aligned with the Pfam00657 consensus sequence the parent and/or variant lipid acyltransferase for use in the compositions/methods of the invention have at least one, preferably more than one, preferably more than two, of the following, a GDSx block, a GANDY block, a HPT block. Suitably, the parent and/or variant lipid acyltransferase may have a GDSx block and a GANDY block. Alternatively, the parent and/or variant enzyme may have a GDSx block and a HPT block. Preferably the parent and/or variant enzyme comprises at least a GDSx block.

Preferably, when aligned with the Pfam00657 consensus sequence the parent and/or variant enzyme for use in the compositions/methods of the invention have at least one, preferably more than one, preferably more than two, preferably more than three, preferably more than four, preferably more than five, preferably more than six, preferably more than seven, preferably more than eight, preferably more than nine, preferably more than ten, preferably more than eleven, preferably more than twelve, preferably more than thirteen, preferably more than fourteen, of the following amino acid residues when compared to the reference A. hydrophilia polypeptide sequence, namely SEQ ID No. 26: 28hid, 29hid, 30hid, 31hid, 32gly, 33Asp, 34Ser, 35hid, 130hid, 131 Gly, 132Hid, 133Asn, 134Asp, 135hid, 309His

The pfam00657 GDSX domain is a unique identifier which distinguishes proteins possessing this domain from other enzymes.

The pfam00657 consensus sequence is presented in FIG. 1 as SEQ ID No. 1. This is derived from the identification of the pfam family 00657, database version 6, which may also be referred to as pfam00657.6 herein.

The consensus sequence may be updated by using further releases of the pfam database. For example, FIGS. 33 and 34 show the pfam alignment of family 00657, from database version 11, which may also be referred to as pfam00657.11 herein.

The presence of the GDSx, GANDY and HPT blocks are found in the pfam family 00657 from both releases of the database. Future releases of the pfam database can be used to identify the pfam family 00657.

Preferably, the parent lipid acyltransferase enzyme according to the present invention may be characterised using the following criteria:

-   -   (i) the enzyme possesses acyl transferase activity which may be         defined as ester transfer activity whereby the acyl part of an         original ester bond of a lipid acyl donor is transferred to acyl         acceptor to form a new ester;     -   (ii) the enzyme comprises the amino acid sequence motif GDSX,         wherein X is one or more of the following amino acid residues L,         A, V, I, F, Y, H, Q, T, N, M or S.;     -   (iii) the enzyme comprises His-309 or comprises a histidine         residue at a position corresponding to His-309 in the Aeromonas         hydrophila lipolytic enzyme shown in FIG. 2 (SEQ ID No. 2 or SEQ         ID No. 26).

Preferably, the amino acid residue of the GDSX motif is L.

In SEQ ID No. 26 the first 18 amino acid residues form a signal sequence. His-309 of the full length sequence, that is the protein including the signal sequence, equates to His-291 of the mature part of the protein, i.e. the sequence without the signal sequence.

Preferably, the parent lipid acyltransferase enzyme according to the present invention comprises the following catalytic triad: Ser-16, Asp-116 and His-291 or comprises a serine residue, an aspartic acid residue and a histidine residue, respectively, at positions corresponding to Ser-16, Asp-116 and His-291 in the Aeromonas hydrophila lipolytic enzyme shown in FIG. 2 (SEQ ID No. 2) or at positions corresponding to Ser-34, Asp-134 and His-309 of the full length sequence shown in FIG. 28 (SEQ ID No. 26). As stated above, in the sequence shown in SEQ ID No. 26 the first 18 amino acid residues form a signal sequence. Ser-34, Asp-134 and His-309 of the full length sequence, that is the protein including the signal sequence, equate to Ser-16, Asp-116 and His-291 of the mature part of the protein, i.e. the sequence without the signal sequence. In the pfam00657 consensus sequence, as given in FIG. 1 (SEQ ID No. 1) the active site residues correspond to Ser-7, Asp-157 and His-348.

Preferably, the parent lipid acyltransferase enzyme according to the present invention may be characterised using the following criteria:

-   -   (i) the enzyme possesses acyl transferase activity which may be         defined as ester transfer activity whereby the acyl part of an         original ester bond of a first lipid acyl donor is transferred         to an acyl acceptor to form a new ester; and     -   (ii) the enzyme comprises at least Gly-14, Asp-15, Ser-16,         Asp-116 and His-191 at positions corresponding to Aeromonas         hydrophila enzyme in FIG. 2 (SEQ ID No. 2) which is equivalent         to positions Gly-32, Asp-33, Ser-34, Asp-134 and His-309,         respectively, in FIG. 28 (SEQ ID No. 26).

Suitably, the parent lipid acyltransferase enzyme according to the present invention may be obtainable, preferably obtained, from organisms from one or more of the following genera: Aeromonas, Corynebacterium, Novosphingobium, Termobifida, Streptomyces, Saccharomyces, Lactococcus, Mycobacterium, Streptococcus, Lactobacillus, Desulfitobacterium, Bacillus, Campylobacter, Vibrionaceae, Xylella, Sulfolobus, Aspergillus, Schizosaccharomyces, Listeria, Neisseria, Mesorhizobium, Ralstonia, Xanthomonas and Candida.

Suitably, the parent lipid acyltransferase enzyme according to the present invention may be obtainable, preferably obtained, from one or more of the following organisms: Aeromonas hydrophila, Aeromonas salmonicida, Streptomyces coelicolor, Streptomyces rimosus, Mycobacterium, Streptococcus pyogenes, Lactococcus lactis, Streptococcus pyogenes, Streptococcus thermophilus, Lactobacillus helveticus, Desulfitobacterium dehalogenans, Bacillus sp, Campylobacter jejuni, Vibrionaceae, Xylella fastidiosa, Sulfolobus solfataricus, Saccharomyces cerevisiae, Aspergillus terreus, Schizosaccharomyces pombe, Listeria innocua, Listeria monocytogenes, Neisseria meningitidis, Mesorhizobium loti, Ralstonia solanacearum, Xanthomonas campestris, Xanthomonas axonopodis, Corynebacterium efficens, Novosphingobium aromaticivorans, Termobifida fusca and Candida parapsilosis.

In one aspect, preferably the parent lipid acyltransferase enzyme according to the present invention is obtainable, preferably obtained, from one or more of Aeromonas hydrophila or Aeromonas salmonicida.

In one aspect, the parent lipid acyltransferase according to the present invention may be a lecithin:cholesterol acyltransferases (LCAT) or variant thereof (for example a variant made by molecular evolution)

Suitable LCATs are known in the art and may be obtainable from one or more of the following organisms for example: mammals, rat, mice, chickens, Drosophila melanogaster, plants, including Arabidopsis and Oryza sativa, nematodes, fungi and yeast.

Preferably, when carrying out a method according to the present invention the product (i.e. foodstuff) is produced without increasing or substantially increasing the free fatty acids in the foodstuff.

The term “transferase” as used herein is interchangeable with the term “lipid acyltransferase”.

Suitably, the lipid acyltransferase as defined herein catalyses one or more of the following reactions: interesterification, transesterification, alcoholysis, hydrolysis.

The term “interesterification” refers to the enzymatic catalysed transfer of acyl groups between a lipid donor and lipid acceptor, wherein the lipid donor is not a free acyl group.

The term “transesterification” as used herein means the enzymatic catalysed transfer of an acyl group from a lipid donor (other than a free fatty acid) to an acyl acceptor (other than water).

As used herein, the term “alcoholysis” refers to the enzymatic cleavage of a covalent bond of an acid derivative by reaction with an alcohol ROH so that one of the products combines with the H of the alcohol and the other product combines with the OR group of the alcohol.

As used herein, the term “alcohol” refers to an alkyl compound containing a hydroxyl group.

As used herein, the term “hydrolysis” refers to the enzymatic catalysed transfer of an acyl group from a lipid to the OH group of a water molecule. Acyl transfer which results from hydrolysis requires the separation of the water molecule.

The term “without increasing or without substantially increasing the free fatty acids” as used herein means that preferably the lipid acyl transferase according to the present invention has 100% transferase activity (i.e. transfers 100% of the acyl groups from an acyl donor onto the acyl acceptor, with no hydrolytic activity); however, the enzyme may transfer less than 100% of the acyl groups present in the lipid acyl donor to the acyl acceptor. In which case, preferably the acyltransferase activity accounts for at least 5%, more preferably at least 10%, more preferably at least 20%, more preferably at least 30%, more preferably at least 40%, more preferably 50%, more preferably at least 60%, more preferably at least 70%, more preferably at least 80%, more preferably at least 90% and more preferably at least 98% of the total enzyme activity. The % transferase activity (i.e. the transferase activity as a percentage of the total enzymatic activity) may be determined by the following protocol:

Protocol for the Determination of % Acyltransferase Activity:

A foodstuff to which a lipid acyltransferase according to the present invention has been added may be extracted following the enzymatic reaction with CHCl₃:CH₃OH 2:1 and the organic phase containing the lipid material is isolated and analysed by GLC according to the procedure detailed hereinbelow. From the GLC analysis (and if necessary HPLC analysis) the amount of free fatty acids and one or more of sterol/stanol esters; carbohydrate esters, protein esters; diglycerides; or monoglycerides are determined. A control foodstuff to which no enzyme according to the present invention has been added, is analysed in the same way.

Calculation:

From the results of the GLC (and optionally HPLC analyses) the increase in free fatty acids and sterol/stanol esters and/or carbohydrate esters and/or protein esters and/or diglycerides and/or monoglycerides can be calculated: Δ % fatty acid=% Fatty acid(enzyme)−% fatty acid(control); Mv fatty acid=average molecular weight of the fatty acids; A=Δ % sterol ester/Mv sterol ester (where Δ % sterol ester=% sterol/stanol ester(enzyme)−% sterol/stanol ester(control) and Mv sterol ester=average molecular weight of the sterol/stanol esters)−applicable where the acyl acceptor is a sterol and/or stanol; B=Δ % carbohydrate ester/Mv carbohydrate ester (where Δ % carbohydrate ester=% carbohydrate ester(enzyme)−% carbohydrate ester(control) and Mv carbohydrate ester=average molecular weight of the carbohydrate ester)−applicable where the acyl acceptor is a carbohydrate; C=Δ % protein ester/Mv protein ester (where Δ % protein ester=% protein ester(enzyme)−% protein ester(control) and Mv protein ester=average molecular weight of the protein ester)−applicable where the acyl acceptor is a protein; and D=absolute value of diglyceride and/or monoglyceride/Mv di/monoglyceride (where Δ % diglyceride and/or monoglyceride=% diglyceride and/or monoglyceride (enzyme)−% diglyceride and/or monoglyceride (control) and Mv di/monoglyceride=average molecular weight of the diglyceride and/or monoglyceride)−applicable where the acyl acceptor is glycerol.

The transferase activity is calculated as a percentage of the total enzymatic activity: ${\%\quad{transferase}\quad{activity}} = {{\frac{A^{*} + B^{*} + C^{*} + {D^{*} \times 100}}{A^{*} + B^{*} + C^{*} + D^{*} + {\Delta\quad\%\quad{fatty}\quad{{acid}/\left( {{Mv}\quad{fatty}\quad{acid}} \right)}}}.{\,^{*}{- {delete}}}}\quad{as}\quad{{appropriate}.}}$

The amino acids which fall within the terms “non-polar”, “polar—uncharged”, “polar—charged” are given in the table below, as are the amino acids falling within the terms “aliphatic” and “aromatic”. The term “polar” refers to both “polar—uncharged” and “polar—charged” amino acids. ALIPHATIC Non-polar G A P I L V Polar - uncharged C S T M N Q Polar - charged D E K R AROMATIC H F W Y

GLC Analysis

Perkin Elmer Autosystem 9000 Capillary Gas Chromatograph equipped with WCOT fused silica column 12.5 m×0.25 mm ID×0.1μ film thickness 5% phenyl-methyl-silicone (CP Sil 8 CB from Chrompack).

Carrier gas: Helium.

Injector. PSSI cold split injection (initial temp 50° C. heated to 385° C.), volume 1.0 μl

Detector FID: 395° C. Oven program: 1 2 3 Oven temperature, 0° C. 90 280 350 Isothermal, time, min. 1 0 10 Temperature rate, ° C./min. 15 4

Sample preparation: 30 mg of sample was dissolved in 9 ml Heptane:Pyridin, 2:1 containing internal standard heptadecane, 0.5 mg/ml. 300 μl sample solution was transferred to a crimp vial, 300 μl MSTFA (N-Methyl-N-trimethylsilyl-trifluoraceamid) was added and reacted for 20 minutes at 60° C.

Calculation: Response factors for mono-di-triglycerides and free fatty acid were determined from Standard 2 (mono-di-triglyceride), for Cholesterol, Cholesteryl palmitate and Cholesteryl stearate the response factors were determined from pure reference material (weighing for pure material 10 mg).

Advantages

Variants transferases of the present invention have one or more of the following advantageous properties compared with the parent enzyme:

-   -   i) an increased activity on polar lipids and/or an increased         activity on polar lipids compared to triglycerides.     -   ii) an increased activity on galactolipids (glycolipids), such         as one or more of digalactosyl diglyceride (DGDG) and/or         monogalactosyl diglyceride (MGDG).     -   iii) an increased ratio of activity on galactolipids         (glycolipids) compared to either phospholipids and/or         triglycerides

Preferably variants transferases of the invention have increased activity on digalactosyl diglyceride (DGDG) and/or monogalactosyl diglyceride (MGDG).

The variants transferases of the invention may also have an increased activity on triglycerides.

The variants transferases of the invention may also have an increased activity on phospholipids, such as lecithin, including phosphatidyl choline.

Variants transferases of the present invention may have decreased activity on triglycerides, and/or monoglycerides and/or diglycerides.

The term polar lipid refers to the polar lipids usually found in a dough, preferably galactolipids and phospholipids.

When used in preparation of a dough or baked product the variant transferase of the invention may result in one or more of the following unexpected technical effects in dough and/or baked products: an improved specific volume of either the dough or the baked products (for example of bread and/or of cake); an improved dough stability; an improved crust score (for example a thinner and/or crispier bread crust), an improved crumb score (for example a more homogenous crumb distribution and/or a finer crumb structure and/or a softer crumb); an improved appearance (for example a smooth surface without blisters or holes or substantially without blisters or holes); a reduced staling; an enhanced softness; an improved odour; an improved taste.

Isolated

In one aspect, preferably the polypeptide or protein for use in the present invention is in an isolated form. The term “isolated” means that the sequence is at least substantially free from at least one other component with which the sequence is naturally associated in nature and as found in nature.

Purified

In one aspect, preferably the polypeptide or protein for use in the present invention is in a purified form. The term “purified” means that the sequence is in a relatively pure state—e.g. at least about 51% pure, or at least about 75%, or at least about 80%, or at least about 90% pure, or at least about 95% pure or at least about 98% pure.

CLONING A NUCLEOTIDE SEQUENCE ENCODING A POLYPEPTIDE ACCORDING TO THE PRESENT INVENTION

A nucleotide sequence encoding either a polypeptide which has the specific properties as defined herein or a polypeptide which is suitable for modification may be isolated from any cell or organism producing said polypeptide. Various methods are well known within the art for the isolation of nucleotide sequences.

For example, a genomic DNA and/or cDNA library may be constructed using chromosomal DNA or messenger RNA from the organism producing the polypeptide. If the amino acid sequence of the polypeptide is known, labelled oligonucleotide probes may be synthesised and used to identify polypeptide-encoding clones from the genomic library prepared from the organism. Alternatively, a labelled oligonucleotide probe containing sequences homologous to another known polypeptide gene could be used to identify polypeptide-encoding clones. In the latter case, hybridisation and washing conditions of lower stringency are used.

Alternatively, polypeptide-encoding clones could be identified by inserting fragments of genomic DNA into an expression vector, such as a plasmid, transforming enzyme-negative bacteria with the resulting genomic DNA library, and then plating the transformed bacteria onto agar containing an enzyme inhibited by the polypeptide, thereby allowing clones expressing the polypeptide to be identified.

In a yet further alternative, the nucleotide sequence encoding the polypeptide may be prepared synthetically by established standard methods, e.g. the phosphoroamidite method described by Beucage S. L. et al (1981) Tetrahedron Letters 22, p 1859-1869, or the method described by Matthes et al (1984) EMBO J. 3, p 801-805. In the phosphoroamidite method, oligonucleotides are synthesised, e.g. in an automatic DNA synthesiser, purified, annealed, ligated and cloned in appropriate vectors.

The nucleotide sequence may be of mixed genomic and synthetic origin, mixed synthetic and cDNA origin, or mixed genomic and cDNA origin, prepared by ligating fragments of synthetic, genomic or cDNA origin (as appropriate) in accordance with standard techniques. Each ligated fragment corresponds to various parts of the entire nucleotide sequence. The DNA sequence may also be prepared by polymerase chain reaction (PCR) using specific primers, for instance as described in U.S. Pat. No. 4,683,202 or in Saiki R K et al (Science (1988) 239, pp 487-491).

Nucleotide Sequences

The present invention also encompasses nucleotide sequences encoding polypeptides having the specific properties as defined herein. The term “nucleotide sequence” as used herein refers to an oligonucleotide sequence or polynucleotide sequence, and variant, homologues, fragments and derivatives thereof (such as portions thereof). The nucleotide sequence may be of genomic or synthetic or recombinant origin, which may be double-stranded or single-stranded whether representing the sense or antisense strand.

The term “nucleotide sequence” in relation to the present invention includes genomic DNA, cDNA, synthetic DNA, and RNA. Preferably it means DNA, more preferably cDNA for the coding sequence.

In a preferred embodiment, the nucleotide sequence per se encoding a polypeptide having the specific properties as defined herein does not cover the native nucleotide sequence in its natural environment when it is linked to its naturally associated sequence(s) that is/are also in its/their natural environment. For ease of reference, we shall call this preferred embodiment the “non-native nucleotide sequence”. In this regard, the term “native nucleotide sequence” means an entire nucleotide sequence that is in its native environment and when operatively linked to an entire promoter with which it is naturally associated, which promoter is also in its native environment. Thus, the polypeptide of the present invention can be expressed by a nucleotide sequence in its native organism but wherein the nucleotide sequence is not under the control of the promoter with which it is naturally associated within that organism.

Preferably the polypeptide is not a native polypeptide. In this regard, the term “native polypeptide” means an entire polypeptide that is in its native environment and when it has been expressed by its native nucleotide sequence.

Typically, the nucleotide sequence encoding polypeptides having the specific properties as defined herein is prepared using recombinant DNA techniques (i.e. recombinant DNA). However, in an alternative embodiment of the invention, the nucleotide sequence could be synthesised, in whole or in part, using chemical methods well known in the art (see Caruthers M H et al (1980) Nuc Acids Res Symp Ser 215-23 and Horn T et al (1980) Nuc Acids Res Symp Ser 225-232).

Molecular Evolution

Once an enzyme-encoding nucleotide sequence has been isolated, or a putative enzyme-encoding nucleotide sequence has been identified, it may be desirable to modify the selected nucleotide sequence, for example it may be desirable to mutate the sequence in order to prepare an enzyme in accordance with the present invention.

Mutations may be introduced using synthetic oligonucleotides. These oligonucleotides contain nucleotide sequences flanking the desired mutation sites.

A suitable method is disclosed in Morinaga et al (Biotechnology (1984) 2, p 646-649). Another method of introducing mutations into enzyme-encoding nucleotide sequences is described in Nelson and Long (Analytical Biochemistry (1989), 180, p 147-151).

Instead of site directed mutagenesis, such as described above, one can introduce mutations randomly for instance using a commercial kit such as the GeneMorph PCR mutagenesis kit from Stratagene, or the Diversify PCR random mutagenesis kit from Clontech. EP 0 583 265 refers to methods of optimising PCR based mutagenesis, which can also be combined with the use of mutagenic DNA analogues such as those described in EP 0 866 796. Error prone PCR technologies are suitable for the production of variants of lipid acyl transferases with preferred characterisitics. WO0206457 refers to molecular evolution of lipases.

A third method to obtain novel sequences is to fragment non-identical nucleotide sequences, either by using any number of restriction enzymes or an enzyme such as Dnase I, and reassembling full nucleotide sequences coding for functional proteins. Alternatively one can use one or multiple non-identical nucleotide sequences and introduce mutations during the reassembly of the full nucleotide sequence. DNA shuffling and family shuffling technologies are suitable for the production of variants of lipid acyl transferases with preferred characteristics. Suitable methods for performing ‘shuffling’ can be found in EP0 752 008, EP1 138 763, EP1 103 606. Shuffling can also be combined with other forms of DNA mutagenesis as described in U.S. Pat. No. 6,180,406 and WO 01/34835.

Thus, it is possible to produce numerous site directed or random mutations into a nucleotide sequence, either in vivo or in vitro, and to subsequently screen for improved functionality of the encoded polypeptide by various means. Using in silico and exo mediated recombination methods (see WO 00/58517, U.S. Pat. No. 6,344,328, U.S. Pat. No. 6,361,974), for example, molecular evolution can be performed where the variant produced retains very low homology to known enzymes or proteins. Such variants thereby obtained may have significant structural analogy to known transferase enzymes, but have very low amino acid sequence homology.

As a non-limiting example, In addition, mutations or natural variants of a polynucleotide sequence can be recombined with either the wild type or other mutations or natural variants to produce new variants. Such new variants can also be screened for improved functionality of the encoded polypeptide.

The application of the above-mentioned and similar molecular evolution methods allows the identification and selection of variants of the enzymes of the present invention which have preferred characteristics without any prior knowledge of protein structure or function, and allows the production of non-predictable but beneficial mutations or variants. There are numerous examples of the application of molecular evolution in the art for the optimisation or alteration of enzyme activity, such examples include, but are not limited to one or more of the following: optimised expression and/or activity in a host cell or in vitro, increased enzymatic activity, altered substrate and/or product specificity, increased or decreased enzymatic or structural stability, altered enzymatic activity/specificity in preferred environmental conditions, e.g. temperature, pH, substrate

As will be apparent to a person skilled in the art, using molecular evolution tools an enzyme may be altered to improve the functionality of the enzyme.

Suitably, the lipid acyltransferase used in the invention may be a variant, i.e. may contain at least one amino acid substitution, deletion or addition, when compared to a parental enzyme. Variant enzymes retain at least 25%, 30%, 40%, 50 %, 60%, 70%, 80%, 90%, 95%, 97%, 99% homology with the parent enzyme. Suitable parent enzymes may include any enzyme with esterase or lipase activity. Preferably, the parent enzyme aligns to the pfam00657 consensus sequence.

In a preferable embodiment a variant lipid acyltransferase enzyme retains or incorporates at least one or more of the pfam00657 consensus sequence amino acid residues found in the GDSx, GANDY and HPT blocks.

Enzymes, such as lipases with no or low lipid acyltransferase activity in an aqueous environment may be mutated using molecular evolution tools to introduce or enhance the transferase activity, thereby producing a lipid acyltransferase enzyme with significant transferase activity suitable for use in the compositions and methods of the present invention.

Suitably, the lipid acyltransferase for use in the invention may be a variant with enhanced enzyme activity on polar lipids, preferably phospholipids and/or glycolipids when compared to the parent enzyme. Preferably, such variants also have low or no activity on lyso polar lipids. The enhanced activity on polar lipids, phospholipids and/or glycolipids may be the result of hydrolysis and/or transferase activity or a combination of both.

Variant lipid acyltransferases for use in the invention may have decreased activity on triglycerides, and/or monoglycerides and/or diglycerides compared with the parent enzyme.

Suitably the variant enzyme may have no activity on triglycerides and/or monoglycerides and/or diglycerides.

Alternatively, the variant enzyme for use in the invention may have increased activity on triglycerides, and/or may also have increased activity on one or more of the following, polar lipids, phospholipids, lecithin, phosphatidylcholine, glycolipids, digalactosyl monoglyceride, monogalactosyl monoglyceride.

Variants of lipid acyltransferases are known, and one or more of such variants may be suitable for use in the methods and uses according to the present invention and/or in the enzyme compositions according to the present invention. By way of example only, variants of lipid acyltransferases are described in the following references may be used in accordance with the present invention: Hilton & Buckley J Biol. Chem. Jan. 15, 1991: 266 (2): 997-1000; Robertson et al J. Biol. Chem. Jan. 21, 1994; 269(3):2146-50; Brumlik et al J. Bacteriol 1996 April; 178 (7): 2060-4; Peelman et al Protein Sci. 1998 March; 7(3):587-99.

Amino Acid Sequences

The present invention also encompasses amino acid sequences of polypeptides having the specific properties as defined herein.

As used herein, the term “amino acid sequence” is synonymous with the term “polypeptide” and/or the term “protein”. In some instances, the term “amino acid sequence” is synonymous with the term “peptide”.

The amino acid sequence may be prepared/isolated from a suitable source, or it may be made synthetically or it may be prepared by use of recombinant DNA techniques.

Suitably, the amino acid sequences may be obtained from the isolated polypeptides taught herein by standard techniques.

One suitable method for determining amino acid sequences from isolated polypeptides is as follows:

Purified polypeptide may be freeze-dried and 100 μg of the freeze-dried material may be dissolved in 50 μl of a mixture of 8 M urea and 0.4 M ammonium hydrogen carbonate, pH 8.4. The dissolved protein may be denatured and reduced for 15 minutes at 50° C. following overlay with nitrogen and addition of 5 μl of 45 mM dithiothreitol. After cooling to room temperature, 5 μl of 100 mM iodoacetamide may be added for the cysteine residues to be derivatized for 15 minutes at room temperature in the dark under nitrogen.

135 μl of water and 5 μg of endoproteinase Lys-C in 5 μl of water may be added to the above reaction mixture and the digestion may be carried out at 37° C. under nitrogen for 24 hours.

The resulting peptides may be separated by reverse phase HPLC on a VYDAC C18 column (0.46×15 cm; 10 μm; The Separation Group, California, USA) using solvent A: 0.1% TFA in water and solvent B: 0.1% TFA in acetonitrile. Selected peptides may be re-chromatographed on a Develosil C18 column using the same solvent system, prior to N-terminal sequencing. Sequencing may be done using an Applied Biosystems 476A sequencer using pulsed liquid fast cycles according to the manufacturer's instructions (Applied Biosystems, California, USA).

Sequence Identity or Sequence Homology

The present invention also encompasses the use of sequences having a degree of sequence identity or sequence homology with amino acid sequence(s) of a polypeptide having the specific properties defined herein or of any nucleotide sequence encoding such a polypeptide (hereinafter referred to as a “homologous sequence(s)”). Here, the term “homologue” means an entity having a certain homology with the subject amino acid sequences and the subject nucleotide sequences. Here, the term “homology” can be equated with “identity”.

The homologous amino acid sequence and/or nucleotide sequence should provide and/or encode a polypeptide which retains the functional activity and/or enhances the activity of the enzyme.

In the present context, a homologous sequence is taken to include an amino acid sequence which may be at least 75, 85 or 90% identical, preferably at least 95 or 98% identical to the subject sequence. Typically, the homologues will comprise the same active sites etc. as the subject amino acid sequence. Although homology can also be considered in terms of similarity (i.e. amino acid residues having similar chemical properties/functions), in the context of the present invention it is preferred to express homology in terms of sequence identity.

In the present context, a homologous sequence is taken to include a nucleotide sequence which may be at least 75, 85 or 90% identical, preferably at least 95 or 98% identical to a nucleotide sequence encoding a polypeptide of the present invention (the subject sequence). Typically, the homologues will comprise the same sequences that code for the active sites etc. as the subject sequence. Although homology can also be considered in terms of similarity (i.e. amino acid residues having similar chemical properties/functions), in the context of the present invention it is preferred to express homology in terms of sequence identity.

Homology comparisons can be conducted by eye, or more usually, with the aid of readily available sequence comparison programs. These commercially available computer programs can calculate % homology between two or more sequences.

% homology may be calculated over contiguous sequences, i.e. one sequence is aligned with the other sequence and each amino acid in one sequence is directly compared with the corresponding amino acid in the other sequence, one residue at a time. This is called an “ungapped” alignment. Typically, such ungapped alignments are performed only over a relatively short number of residues.

Although this is a very simple and consistent method, it fails to take into consideration that, for example, in an otherwise identical pair of sequences, one insertion or deletion will cause the following amino acid residues to be put out of alignment, thus potentially resulting in a large reduction in % homology when a global alignment is performed. Consequently, most sequence comparison methods are designed to produce optimal alignments that take into consideration possible insertions and deletions without penalising unduly the overall homology score. This is achieved by inserting “gaps” in the sequence alignment to try to maximise local homology.

However, these more complex methods assign “gap penalties” to each gap that occurs in the alignment so that, for the same number of identical amino acids, a sequence alignment with as few gaps as possible—reflecting higher relatedness between the two compared sequences—will achieve a higher score than one with many gaps. “Affine gap costs” are typically used that charge a relatively high cost for the existence of a gap and a smaller penalty for each subsequent residue in the gap. This is the most commonly used gap scoring system. High gap penalties will of course produce optimised alignments with fewer gaps. Most alignment programs allow the gap penalties to be modified. However, it is preferred to use the default values when using such software for sequence comparisons. For example when using the GCG Wisconsin Bestfit package the default gap penalty for amino acid sequences is −12 for a gap and −4 for each extension.

Calculation of maximum % homology therefore firstly requires the production of an optimal alignment, taking into consideration gap penalties. A suitable computer program for carrying out such an alignment is the GCG Wisconsin Bestfit package (Devereux et al 1984 Nuc. Acids Research 12 p 387). Examples of other software that can perform sequence comparisons include, but are not limited to, the BLAST package (see Ausubel et al 1999 Short Protocols in Molecular Biology, 4^(th) Ed—Chapter 18), FASTA (Altschul et al 1990 J. Mol. Biol. 403-410) and the GENEWORKS suite of comparison tools. Both BLAST and FASTA are available for offline and online searching (see Ausubel et al 1999, pages 7-58 to 7-60). However, for some applications, it is preferred to use the GCG Bestfit program. A new tool, called BLAST 2 Sequences is also available for comparing protein and nucleotide sequence (see FEMS Microbiol Lett 1999 174(2): 247-50; FEMS Microbiol Lett 1999 177(1): 187-8 and tatiana@ncbi.nlm.nih.gov).

Although the final % homology can be measured in terms of identity, the alignment process itself is typically not based on an all-or-nothing pair comparison. Instead, a scaled similarity score matrix is generally used that assigns scores to each pairwise comparison based on chemical similarity or evolutionary distance. An example of such a matrix commonly used is the BLOSUM62 matrix—the default matrix for the BLAST suite of programs. GCG Wisconsin programs generally use either the public default values or a custom symbol comparison table if supplied (see user manual for further details). For some applications, it is preferred to use the public default values for the GCG package, or in the case of other software, the default matrix, such as BLOSUM62.

Alternatively, percentage homologies may be calculated using the multiple alignment feature in DNASIS™ (Hitachi Software), based on an algorithm, analogous to CLUSTAL (Higgins D G & Sharp P M (1988), Gene 73(1), 237-244).

Once the software has produced an optimal alignment, it is possible to calculate % homology, preferably % sequence identity. The software typically does this as part of the sequence comparison and generates a numerical result.

The sequences may also have deletions, insertions or substitutions of amino acid residues which produce a silent change and result in a functionally equivalent substance. Deliberate amino acid substitutions may be made on the basis of similarity in polarity, charge, solubility, hydrophobicity, hydrophilicity, and/or the amphipathic nature of the residues as long as the secondary binding activity of the substance is retained. For example, negatively charged amino acids include aspartic acid and glutamic acid; positively charged amino acids include lysine and arginine; and amino acids with uncharged polar head groups having similar hydrophilicity values include leucine, isoleucine, valine, glycine, alanine, asparagine, glutamine, serine, threonine, phenylalanine, and tyrosine.

Conservative substitutions may be made, for example according to the Table below. Amino acids in the same block in the second column and preferably in the same line in the third column may be substituted for each other: ALIPHATIC Non-polar G A P I L V Polar - uncharged C S T M N Q Polar - charged D E K R AROMATIC H F W Y

The present invention also encompasses homologous substitution (substitution and replacement are both used herein to mean the interchange of an existing amino acid residue, with an alternative residue) that may occur i.e. like-for-like substitution such as basic for basic, acidic for acidic, polar for polar etc. Non-homologous substitution may also occur i.e. from one class of residue to another or alternatively involving the inclusion of unnatural amino acids such as ornithine (hereinafter referred to as Z), diaminobutyric acid ornithine (hereinafter referred to as B), norleucine ornithine (hereinafter referred to as O), pyriylalanine, thienylalanine, naphthylalanine and phenylglycine.

Replacements may also be made by unnatural amino acids.

Variant amino acid sequences may include suitable spacer groups that may be inserted between any two amino acid residues of the sequence including alkyl groups such as methyl, ethyl or propyl groups in addition to amino acid spacers such as glycine or β-alanine residues. A further form of variation, involves the presence of one or more amino acid residues in peptoid form, will be well understood by those skilled in the art. For the avoidance of doubt, “the peptoid form” is used to refer to variant amino acid residues wherein the α-carbon substituent group is on the residue's nitrogen atom rather than the α-carbon. Processes for preparing peptides in the peptoid form are known in the art, for example Simon R J et al., PNAS (1992) 89(20), 9367-9371 and Horwell D C, Trends Biotechnol. (1995) 13(4), 132-134.

Nucleotide sequences for use in the present invention or encoding a polypeptide having the specific properties defined herein may include within them synthetic or modified nucleotides. A number of different types of modification to oligonucleotides are known in the art. These include methylphosphonate and phosphorothioate backbones and/or the addition of acridine or polylysine chains at the 3′ and/or 5′ ends of the molecule. For the purposes of the present invention, it is to be understood that the nucleotide sequences described herein may be modified by any method available in the art. Such modifications may be carried out in order to enhance the in vivo activity or life span of nucleotide sequences.

The present invention also encompasses the use of nucleotide sequences that are complementary to the sequences discussed herein, or any derivative, fragment or derivative thereof. If the sequence is complementary to a fragment thereof then that sequence can be used as a probe to identify similar coding sequences in other organisms etc.

Polynucleotides which are not 100% homologous to the sequences of the present invention but fall within the scope of the invention can be obtained in a number of ways. Other variants of the sequences described herein may be obtained for example by probing DNA libraries made from a range of individuals, for example individuals from different populations. In addition, other viral/bacterial, or cellular homologues particularly cellular homologues found in mammalian cells (e.g. rat, mouse, bovine and primate cells), may be obtained and such homologues and fragments thereof in general will be capable of selectively hybridising to the sequences shown in the sequence listing herein. Such sequences may be obtained by probing cDNA libraries made from or genomic DNA libraries from other animal species, and probing such libraries with probes comprising all or part of any one of the sequences in the attached sequence listings under conditions of medium to high stringency. Similar considerations apply to obtaining species homologues and allelic variants of the polypeptide or nucleotide sequences of the invention.

Variants and strain/species homologues may also be obtained using degenerate PCR which will use primers designed to target sequences within the variants and homologues encoding conserved amino acid sequences within the sequences of the present invention. Conserved sequences can be predicted, for example, by aligning the amino acid sequences from several variants/homologues. Sequence alignments can be performed using computer software known in the art. For example the GCG Wisconsin PileUp program is widely used.

The primers used in degenerate PCR will contain one or more degenerate positions and will be used at stringency conditions lower than those used for cloning sequences with single sequence primers against known sequences.

Alternatively, such polynucleotides may be obtained by site directed mutagenesis of characterised sequences. This may be useful where for example silent codon sequence changes are required to optimise codon preferences for a particular host cell in which the polynucleotide sequences are being expressed. Other sequence changes may be desired in order to introduce restriction polypeptide recognition sites, or to alter the property or function of the polypeptides encoded by the polynucleotides.

Polynucleotides (nucleotide sequences) of the invention may be used to produce a primer, e.g. a PCR primer, a primer for an alternative amplification reaction, a probe e.g. labelled with a revealing label by conventional means using radioactive or non-radioactive labels, or the polynucleotides may be cloned into vectors. Such primers, probes and other fragments will be at least 15, preferably at least 20, for example at least 25, 30 or 40 nucleotides in length, and are also encompassed by the term polynucleotides of the invention as used herein.

Polynucleotides such as DNA polynucleotides and probes according to the invention may be produced recombinantly, synthetically, or by any means available to those of skill in the art. They may also be cloned by standard techniques.

In general, primers will be produced by synthetic means, involving a stepwise manufacture of the desired nucleic acid sequence one nucleotide at a time. Techniques for accomplishing this using automated techniques are readily available in the art.

Longer polynucleotides will generally be produced using recombinant means, for example using a PCR (polymerase chain reaction) cloning techniques. This will involve making a pair of primers (e.g. of about 15 to 30 nucleotides) flanking a region of the lipid targeting sequence which it is desired to clone, bringing the primers into contact with mRNA or cDNA obtained from an animal or human cell, performing a polymerase chain reaction under conditions which bring about amplification of the desired region, isolating the amplified fragment (e.g. by purifying the reaction mixture on an agarose gel) and recovering the amplified DNA. The primers may be designed to contain suitable restriction enzyme recognition sites so that the amplified DNA can be cloned into a suitable cloning vector.

Hybridisation

The present invention also encompasses sequences that are complementary to the sequences of the present invention or sequences that are capable of hybridising either to the sequences of the present invention or to sequences that are complementary thereto.

The term “hybridisation” as used herein shall include “the process by which a strand of nucleic acid joins with a complementary strand through base pairing” as well as the process of amplification as carried out in polymerase chain reaction (PCR) technologies.

The present invention also encompasses the use of nucleotide sequences that are capable of hybridising to the sequences that are complementary to the subject sequences discussed herein, or any derivative, fragment or derivative thereof.

The present invention also encompasses sequences that are complementary to sequences that are capable of hybridising to the nucleotide sequences discussed herein.

Hybridisation conditions are based on the melting temperature (Tm) of the nucleotide binding complex, as taught in Berger and Kimmel (1987, Guide to Molecular Cloning Techniques, Methods in Enzymology, Vol. 152, Academic Press, San Diego Calif.), and confer a defined “stringency” as explained below.

Maximum stringency typically occurs at about Tm-5° C. (5° C. below the Tm of the probe); high stringency at about 5° C. to 10° C. below Tm; intermediate stringency at about 10° C. to 20° C. below Tm; and low stringency at about 20° C. to 25° C. below Tm. As will be understood by those of skill in the art, a maximum stringency hybridisation can be used to identify or detect identical nucleotide sequences while an intermediate (or low) stringency hybridisation can be used to identify or detect similar or related polynucleotide sequences.

Preferably, the present invention encompasses sequences that are complementary to sequences that are capable of hybridising under high stringency conditions or intermediate stringency conditions to nucleotide sequences encoding polypeptides having the specific properties as defined herein.

More preferably, the present invention encompasses sequences that are complementary to sequences that are capable of hybridising under high stringent conditions (e.g. 65° C. and 0.1×SSC {1×SSC=0.15 M NaCl, 0.015 M Na-citrate pH 7.0}) to nucleotide sequences encoding polypeptides having the specific properties as defined herein.

The present invention also relates to nucleotide sequences that can hybridise to the nucleotide sequences discussed herein (including complementary sequences of those discussed herein).

The present invention also relates to nucleotide sequences that are complementary to sequences that can hybridise to the nucleotide sequences discussed herein (including complementary sequences of those discussed herein).

Also included within the scope of the present invention are polynucleotide sequences that are capable of hybridising to the nucleotide sequences discussed herein under conditions of intermediate to maximal stringency.

In a preferred aspect, the present invention covers nucleotide sequences that can hybridise to the nucleotide sequences discussed herein, or the complement thereof, under stringent conditions (e.g. 50° C. and 0.2×SSC).

In a more preferred aspect, the present invention covers nucleotide sequences that can hybridise to the nucleotide sequences discussed herein, or the complement thereof, under high stringent conditions (e.g. 65° C. and 0.1×SSC).

Expression of Polypeptides

A nucleotide sequence for use in the present invention or for encoding a polypeptide having the specific properties as defined herein can be incorporated into a recombinant replicable vector. The vector may be used to replicate and express the nucleotide sequence, in polypeptide form, in and/or from a compatible host cell. Expression may be controlled using control sequences which include promoters/enhancers and other expression regulation signals. Prokaryotic promoters and promoters functional in eukaryotic cells may be used. Tissue specific or stimuli specific promoters may be used. Chimeric promoters may also be used comprising sequence elements from two or more different promoters described above.

The polypeptide produced by a host recombinant cell by expression of the nucleotide sequence may be secreted or may be contained intracellularly depending on the sequence and/or the vector used. The coding sequences can be designed with signal sequences which direct secretion of the substance coding sequences through a particular prokaryotic or eukaryotic cell membrane.

Expression Vector

The term “expression vector” means a construct capable of in vivo or in vitro expression.

Preferably, the expression vector is incorporated in the genome of the organism. The term “incorporated” preferably covers stable incorporation into the genome.

The nucleotide sequence of the present invention or coding for a polypeptide having the specific properties as defined herein may be present in a vector, in which the nucleotide sequence is operably linked to regulatory sequences such that the regulatory sequences are capable of providing the expression of the nucleotide sequence by a suitable host organism, i.e. the vector is an expression vector.

The vectors of the present invention may be transformed into a suitable host cell as described below to provide for expression of a polypeptide having the specific properties as defined herein.

The choice of vector, e.g. plasmid, cosmid, virus or phage vector, will often depend on the host cell into which it is to be introduced.

The vectors may contain one or more selectable marker genes—such as a gene which confers antibiotic resistance e.g. ampicillin, kanamycin, chloramphenicol or tetracyclin resistance. Alternatively, the selection may be accomplished by co-transformation (as described in WO91/17243).

Vectors may be used in vitro, for example for the production of RNA or used to transfect or transform a host cell.

Thus, in a further embodiment, the invention provides a method of making nucleotide sequences of the present invention or nucleotide sequences encoding polypeptides having the specific properties as defined herein by introducing a nucleotide sequence into a replicable vector, introducing the vector into a compatible host cell, and growing the host cell under conditions which bring about replication of the vector.

The vector may further comprise a nucleotide sequence enabling the vector to replicate in the host cell in question. Examples of such sequences are the origins of replication of plasmids pUC19, pACYC177, pUB110, pE194, pAMB1 and pIJ702.

Regulatory Sequences

In some applications, a nucleotide sequence for use in the present invention or a nucleotide sequence encoding a polypeptide having the specific properties as defined herein may be operably linked to a regulatory sequence which is capable of providing for the expression of the nucleotide sequence, such as by the chosen host cell. By way of example, the present invention covers a vector comprising the nucleotide sequence of the present invention operably linked to such a regulatory sequence, i.e. the vector is an expression vector.

The term “operably linked” refers to a juxtaposition wherein the components described are in a relationship permitting them to function in their intended manner. A regulatory sequence “operably linked” to a coding sequence is ligated in such a way that expression of the coding sequence is achieved under conditions compatible with the control sequences.

The term “regulatory sequences” includes promoters and enhancers and other expression regulation signals.

The term “promoter” is used in the normal sense of the art, e.g. an RNA polymerase binding site.

Enhanced expression of the nucleotide sequence encoding the enzyme having the specific properties as defined herein may also be achieved by the selection of heterologous regulatory regions, e.g. promoter, secretion leader and terminator regions.

Preferably, the nucleotide sequence of the present invention may be operably linked to at least a promoter.

Examples of suitable promoters for directing the transcription of the nucleotide sequence in a bacterial, fungal or yeast host are well known in the art.

Constructs

The term “construct”—which is synonymous with terms such as “conjugate”, “cassette” and “hybrid”—includes a nucleotide sequence encoding a polypeptide having the specific properties as defined herein for use according to the present invention directly or indirectly attached to a promoter. An example of an indirect attachment is the provision of a suitable spacer group such as an intron sequence, such as the Sh1-intron or the ADH intron, intermediate the promoter and the nucleotide sequence of the present invention. The same is true for the term “fused” in relation to the present invention which includes direct or indirect attachment. In some cases, the terms do not cover the natural combination of the nucleotide sequence coding for the protein ordinarily associated with the wild type gene promoter and when they are both in their natural environment.

The construct may even contain or express a marker which allows for the selection of the genetic construct.

For some applications, preferably the construct comprises at least a nucleotide sequence of the present invention or a nucleotide sequence encoding a polypeptide having the specific properties as defined herein operably linked to a promoter.

Host Cells

The term “host cell”—in relation to the present invention includes any cell that comprises either a nucleotide sequence encoding a polypeptide having the specific properties as defined herein or an expression vector as described above and which is used in the recombinant production of a polypeptide having the specific properties as defined herein.

Thus, a further embodiment of the present invention provides host cells transformed or transfected with a nucleotide sequence of the present invention or a nucleotide sequence that expresses a polypeptide having the specific properties as defined herein. The cells will be chosen to be compatible with the said vector and may for example be prokaryotic (for example bacterial), fungal, yeast or plant cells. Preferably, the host cells are not human cells.

Examples of suitable bacterial host organisms are gram negative bacterium or gram positive bacteria.

Depending on the nature of the nucleotide sequence encoding a polypeptide having the specific properties as defined herein, and/or the desirability for further processing of the expressed protein, eukaryotic hosts such as yeasts or other fungi may be preferred. In general, yeast cells are preferred over fungal cells because they are easier to manipulate. However, some proteins are either poorly secreted from the yeast cell, or in some cases are not processed properly (e.g. hyperglycosylation in yeast). In these instances, a different fungal host organism should be selected.

The use of suitable host cells, such as yeast, fungal and plant host cells—may provide for post-translational modifications (e.g. myristoylation, glycosylation, truncation, lapidation and tyrosine, serine or threonine phosphorylation) as may be needed to confer optimal biological activity on recombinant expression products of the present invention.

The host cell may be a protease deficient or protease minus strain.

Organism

The term “organism” in relation to the present invention includes any organism that could comprise a nucleotide sequence according to the present invention or a nucleotide sequence encoding for a polypeptide having the specific properties as defined herein and/or products obtained therefrom.

Suitable organisms may include a prokaryote, fungus, yeast or a plant.

The term “transgenic organism” in relation to the present invention includes any organism that comprises a nucleotide sequence coding for a polypeptide having the specific properties as defined herein and/or the products obtained therefrom, and/or wherein a promoter can allow expression of the nucleotide sequence coding for a polypeptide having the specific properties as defined herein within the organism. Preferably the nucleotide sequence is incorporated in the genome of the organism.

The term “transgenic organism” does not cover native nucleotide coding sequences in their natural environment when they are under the control of their native promoter which is also in its natural environment.

Therefore, the transgenic organism of the present invention includes an organism comprising any one of, or combinations of, a nucleotide sequence coding for a polypeptide having the specific properties as defined herein, constructs as defined herein, vectors as defined herein, plasmids as defined herein, cells as defined herein, or the products thereof. For example the transgenic organism can also comprise a nucleotide sequence coding for a polypeptide having the specific properties as defined herein under the control of a heterologous promoter.

Transformation of Host Cells/Organism

As indicated earlier, the host organism can be a prokaryotic or a eukaryotic organism. Examples of suitable prokaryotic hosts include E. coli and Bacillus subtilis.

Teachings on the transformation of prokaryotic hosts is well documented in the art, for example see Sambrook et al (Molecular Cloning: A Laboratory Manual, 2nd edition, 1989, Cold Spring Harbor Laboratory Press). If a prokaryotic host is used then the nucleotide sequence may need to be suitably modified before transformation—such as by removal of introns.

In another embodiment the transgenic organism can be a yeast.

Filamentous fungi cells may be transformed using various methods known in the art—such as a process involving protoplast formation and transformation of the protoplasts followed by regeneration of the cell wall in a manner known. The use of Aspergillus as a host microorganism is described in EP 0 238 023.

Another host organism can be a plant. A review of the general techniques used for transforming plants may be found in articles by Potrykus (Annu Rev Plant Physiol Plant Mol Biol [1991] 42:205-225) and Christou (Agro-Food-Industry Hi-Tech March/April 1994 17-27). Further teachings on plant transformation may be found in EP-A-0449375.

General teachings on the transformation of fungi, yeasts and plants are presented in following sections.

Transformed Fungus

A host organism may be a fungus—such as a filamentous fungus. Examples of suitable such hosts include any member belonging to the genera Thermomyces, Acremonium, Aspergillus, Penicillium, Mucor, Neurospora, Trichoderma and the like.

Teachings on transforming filamentous fungi are reviewed in U.S. Pat. No. 5,741,665 which states that standard techniques for transformation of filamentous fungi and culturing the fungi are well known in the art. An extensive review of techniques as applied to N. crassa is found, for example in Davis and de Serres, Methods Enzymol (1971) 17A: 79-143.

Further teachings on transforming filamentous fungi are reviewed in U.S. Pat. No. 5,674,707.

In one aspect, the host organism can be of the genus Aspergillus, such as Aspergillus niger.

A transgenic Aspergillus according to the present invention can also be prepared by following, for example, the teachings of Turner G. 1994 (Vectors for genetic manipulation. In: Martinelli S. D., Kinghorn J. R.( Editors) Aspergillus: 50 years on. Progress in industrial microbiology vol 29. Elsevier Amsterdam 1994. pp. 641-666).

Gene expression in filamentous fungi has been reviewed in Punt et al. (2002) Trends Biotechnol 2002 May;20(5):200-6, Archer & Peberdy Crit Rev Biotechnol (1997) 17(4):273-306.

Transformed Yeast

In another embodiment, the transgenic organism can be a yeast.

A review of the principles of heterologous gene expression in yeast are provided in, for example, Methods Mol Biol (1995), 49:341-54, and Curr Opin Biotechnol (1997) October;8(5):554-60

In this regard, yeast—such as the species Saccharomyces cerevisi or Pichia pastoris (see FEMS Microbiol Rev (2000 24(1):45-66), may be used as a vehicle for heterologous gene expression.

A review of the principles of heterologous gene expression in Saccharomyces cerevisiae and secretion of gene products is given by E Hinchcliffe E Kenny (1993, “Yeast as a vehicle for the expression of heterologous genes”, Yeasts, Vol 5, Anthony H Rose and J Stuart Harrison, eds, 2nd edition, Academic Press Ltd.).

For the transformation of yeast, several transformation protocols have been developed. For example, a transgenic Saccharomyces according to the present invention can be prepared by following the teachings of Hinnen et al., (1978, Proceedings of the National Academy of Sciences of the USA 75, 1929); Beggs, J D (1978, Nature, London, 275, 104); and Ito, H et al (1983, J Bacteriology 153, 163-168).

The transformed yeast cells may be selected using various selective markers—such as auxotrophic markers dominant antibiotic resistance markers.

A suitable yeast host organism can be selected from the biotechnologically relevant yeasts species such as, but not limited to, yeast species selected from Pichia spp., Hansenula spp., Kluyveromyces, Yarrowinia spp., Saccharomyces spp., including S. cerevisiae, or Schizosaccharomyce spp. including Schizosaccharomyce pombe.

A strain of the methylotrophic yeast species Pichia pastoris may be used as the host organism.

In one embodiment, the host organism may be a Hansenula species, such as H. polymorpha (as described in WO01/39544).

Transformed Plants/Plant Cells

A host organism suitable for the present invention may be a plant. A review of the general techniques may be found in articles by Potrykus (Annu Rev Plant Physiol Plant Mol Biol [1991] 42:205-225) and Christou (Agro-Food-Industry Hi-Tech March/April 1994 17-27), or in WO01/16308. The transgenic plant may produce enhanced levels of phytosterol esters and phytostanol esters, for example.

Therefore the present invention also relates to a method for the production of a transgenic plant with enhanced levels of phytosterol esters and phytostanol esters, comprising the steps of transforming a plant cell with a lipid acyltransferase as defined herein (in particular with an expression vector or construct comprising a lipid acyltransferase as defined herein), and growing a plant from the transformed plant cell.

Secretion

Often, it is desirable for the polypeptide to be secreted from the expression host into the culture medium from where the enzyme may be more easily recovered. According to the present invention, the secretion leader sequence may be selected on the basis of the desired expression host. Hybrid signal sequences may also be used with the context of the present invention.

Typical examples of heterologous secretion leader sequences are those originating from the fungal amyloglucosidase (AG) gene (glaA—both 18 and 24 amino acid versions e.g. from Aspergillus), the a-factor gene (yeasts e.g. Saccharomyces, Kluyveromyces and Hansenula) or the α-amylase gene (Bacillus).

Detection

A variety of protocols for detecting and measuring the expression of the amino acid sequence are known in the art. Examples include enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA) and fluorescent activated cell sorting (FACS).

A wide variety of labels and conjugation techniques are known by those skilled in the art and can be used in various nucleic and amino acid assays.

A number of companies such as Pharmacia Biotech (Piscataway, N.J.), Promega (Madison, Wis.), and US Biochemical Corp (Cleveland, Ohio) supply commercial kits and protocols for these procedures.

Suitable reporter molecules or labels include those radionuclides, enzymes, fluorescent, chemiluminescent, or chromogenic agents as well as substrates, cofactors, inhibitors, magnetic particles and the like. Patents teaching the use of such labels include U.S. Pat. No. 3,817,837; U.S. Pat. No. 3,850,752; U.S. Pat. No. 3,939,350; U.S. Pat. No. 3,996,345; U.S. Pat. No. 4,277,437; U.S. Pat. No 4,275,149 and U.S. Pat. No. 4,366,241.

Also, recombinant immunoglobulins may be produced as shown in U.S. Pat. No. 4,816,567.

Fusion Proteins

A polypeptide having the specific properties as defined herein may be produced as a fusion protein, for example to aid in extraction and purification thereof. Examples of fusion protein partners include glutathione-S-transferase (GST), 6×His, GAL4 (DNA binding and/or transcriptional activation domains) and β-galactosidase. It may also be convenient to include a proteolytic cleavage site between the fusion protein partner and the protein sequence of interest to allow removal of fusion protein sequences. Preferably the fusion protein will not hinder the activity of the protein sequence.

Gene fusion expression systems in E. coli have been reviewed in Curr. Opin. Biotechnol. (1995) 6(5):501-6.

In another embodiment of the invention, the amino acid sequence of a polypeptide having the specific properties as defined herein may be ligated to a heterologous sequence to encode a fusion protein. For example, for screening of peptide libraries for agents capable of affecting the substance activity, it may be useful to encode a chimeric substance expressing a heterologous epitope that is recognised by a commercially available antibody.

The invention will now be described, by way of example only, with reference to the following Figures and Examples.

FIG. 1 shows a pfam00657 consensus sequence from database version 6 (SEQ ID No. 1);

FIG. 2 shows an amino acid sequence (SEQ ID No. 2) obtained from the organism Aeromonas hydrophila (P10480; GI:121051). This amino acid sequence is a reference enzyme, which may be a parent enzyme in accordance with the present invention;

FIG. 3 shows an amino acid sequence (SEQ ID No. 3) obtained from the organism Aeromonas salmonicida (AAG098404; GI:9964017);

FIG. 4 shows an amino acid sequence (SEQ ID No. 4) obtained from the organism Streptomyces coelicolor A3(2) (Genbank accession number NP_(—)631558);

FIG. 5 shows an amino acid sequence (SEQ ID No. 5) obtained from the organism Streptomyces coelicolor A3(2) (Genbank accession number: CAC42140);

FIG. 6 shows an amino acid sequence (SEQ ID No. 6) obtained from the organism Saccharomyces cerevisiae (Genbank accession number P41734);

FIG. 7 shows an alignment of selected sequences to pfam00657 consensus sequence;

FIG. 8 shows a pairwise alignment of SEQ ID No. 3 with SEQ ID No. 2 showing 93% amino acid sequence identity. The signal sequence is underlined. + denotes differences. The GDSX motif containing the active site serine 16, and the active sites aspartic acid 116 and histidine 291 are highlighted (see shaded regions). Numbers after the amino acid is minus the signal sequence;

FIG. 9 shows a nucleotide sequence (SEQ ID No. 7) encoding a lipid acyl transferase according to the present invention obtained from the organism Aeromonas hydrophila;

FIG. 10 shows a nucleotide sequence (SEQ ID No. 8) encoding a lipid acyl transferase according to the present invention obtained from the organism Aeromonas salmonicida;

FIG. 11 shows a nucleotide sequence (SEQ ID No. 9) encoding a lipid acyl transferase according to the present invention obtained from the organism Streptomyces coelicolor A3(2) (Genbank accession number NC_(—)003888.1:8327480 . . . 8328367);

FIG. 12 shows a nucleotide sequence (SEQ ID No. 10) encoding a lipid acyl transferase according to the present invention obtained from the organism Streptomyces coelicolor A3(2) (Genbank accession number AL939131.1:265480 . . . 266367);

FIG. 13 shows a nucleotide sequence (SEQ ID No. 11) encoding a lipid acyl transferase according to the present invention obtained from the organism Saccharomyces cerevisiae (Genbank accession number Z75034);

FIG. 14 shows an amino acid sequence (SEQ ID No. 12) obtained from the organism Ralstonia (Genbank accession number: AL646052);

FIG. 15 shows a nucleotide sequence (SEQ ID No. 13) encoding a lipid acyl transferase according to the present invention obtained from the organism Ralstonia;

FIG. 16 shows SEQ ID No. 14. Scoe1 NCBI protein accession code CAB39707.1 GI:4539178 conserved hypothetical protein [Streptomyces coelicolor A3(2)];

FIG. 17 shows a nucleotide sequence shown as SEQ ID No. 15 encoding NCBI protein accession code CAB39707.1 GI:4539178 conserved hypothetical protein [Streptomyces coelicolor A3(2)];

FIG. 18 shows an amino acid shown as SEQ ID No. 16. Scoe2 NCBI protein accession code CAC01477.1 GI:9716139 conserved hypothetical protein [Streptomyces coelicolor A3(2)];

FIG. 19 shows a nucleotide sequence shown as SEQ ID No. 17 encoding Scoe2 NCBI protein accession code CAC01477.1 GI:9716139 conserved hypothetical protein [Streptomyces coelicolor A3(2)];

FIG. 20 shows an amino acid sequence (SEQ ID No. 18) Scoe3 NCBI protein accession code CAB88833.1 GI:7635996 putative secreted protein. [Streptomyces coelicolor A3(2)];

FIG. 21 shows a nucleotide sequence shown as SEQ ID No. 19 encoding Scoe3 NCBI protein accession code CAB88833.1 GI:7635996 putative secreted protein. [Streptomyces coelicolor A3(2)];

FIG. 22 shows an amino acid sequence (SEQ ID No. 20) Scoe4 NCBI protein accession code CAB89450.1 GI:7672261 putative secreted protein. [Streptomyces coelicolor A3(2)];

FIG. 23 shows an nucleotide sequence shown as SEQ ID No. 21 encoding Scoe4 NCBI protein accession code CAB89450.1 GI:7672261 putative secreted protein. [Streptomyces coelicolor A3(2)];

FIG. 24 shows an amino acid sequence (SEQ ID No. 22) Scoe5 NCBI protein accession code CAB62724.1 GI:6562793 putative lipoprotein [Streptomyces coelicolor A3(2)];

FIG. 25 shows a nucleotide sequence shown as SEQ ID No. 23, encoding Scoe5 NCBI protein accession code CAB62724.1 GI:6562793 putative lipoprotein [Streptomyces coelicolor A3(2)];

FIG. 26 shows an amino acid sequence (SEQ ID No. 24) Srim1 NCBI protein accession code AAK84028.1 GI:15082088 GDSL-lipase [Streptomyces rimosus];

FIG. 27 shows a nucleotide sequence shown as SEQ ID No. 25 encoding Srim1 NCBI protein accession code AAK84028.1 GI:15082088 GDSL-lipase [Streptomyces rimosus];

FIG. 28 shows an amino acid sequence (SEQ ID No. 26)—a lipid acyl transferase from Aeromonas hydrophila (ATCC #7965);

FIG. 29 shows a nucleotide sequence (SEQ ID No. 27) encoding a lipid acyltransferase from Aeromonas hydrophila (ATCC #7965);

FIG. 30 shows an amino acid sequence (SEQ ID No. 28) of a lipid acyltransferase from Aeromonas salmonicida subsp. Salmonicida (ATCC#14174);

FIG. 31 shows a nucleotide sequence (SEQ ID No. 29) encoding a lipid acyltransferase from Aeromonas salmonicida subsp. Salmonicida (ATCC#14174);

FIG. 32 shows that homologues of the Aeromonas genes can be identified using the basic local alignment search tool service at the National Center for Biotechnology Information, NIH, MD, USA and the completed genome databases. The GDSX motif was used in the database search and a number of sequences/genes potentially encoding enzymes with lipolytic activity were identified. Genes were identified from the genus Streptomyces, Xanthomonas and Ralstonia. As an example below, the Ralstonia solanacearum was aligned to the Aeromonas salmonicida (satA) gene. Pairwise alignment showed 23% identity. The active site serine is present at the amino terminus and the catalytic residues histidine and aspartic acid can be identified;

FIG. 33 shows the Pfam00657.11 [family 00657, database version 11] consensus sequence (hereafter called Pfam consensus) and the alignment of various sequences to the Pfam consensus sequence. The arrows indicate the active site residues, the underlined boxes indicate three of the homology boxes indicated by [Upton C and Buckley J T (1995) Trends Biochem Sci 20; 179-179]. Capital letters in the Pfam consensus indicate conserved residues in many family members. The - symbol indicates a position where the hidden Markov model of the Pfam consensus expected to find a residue but did not, so a gap is inserted. The . symbol indicates a residue without a corresponding residue in the Pfam consensus. The sequences are the amino acid sequences listed in FIGS. 16, 18, 20, 22, 24, 26, 28 and 30.

FIG. 34 shows the Pfam00657.11 [family 00657, database version 11] consensus sequence (hereafter called Pfam consensus) and the alignment of various sequences to the Pfam consensus sequence. The arrows indicate the active site residues, the underlined boxes indicate three of the homology boxes indicated by [Upton C and Buckley J T (1995) Trends Biochem Sci 20; 179-179]. Capital letters in the Pfam consensus indicate conserved residues in many family members. The - symbol indicates a position where the hidden Markov model of the Pfam consensus expected to find a residue but did not, so a gap is inserted. The . symbol indicates a residue without a corresponding residue in the Pfam consensus. The sequences are the amino acid sequences listed in FIGS. 2, 16, 18, 20, 26, 28 and 30. All these proteins were found to be active against lipid substrates.

FIG. 35 shows an amino acid sequence (SEQ ID No. 30) of the fusion construct used for mutagenesis of the Aeromonas hydrophila lipid acyltransferase gene in Example 7. The underlined amino acids is a xylanase signal peptide;

FIG. 36 shows a nucleotide sequence (SEQ ID No. 31) encoding a lipid acyltransferase enzyme from Aeromonas hydrophila including a xylanase signal peptide;

FIG. 37 shows a nucleotide sequence encoding a lipid acyltransferase enzyme from Streptomyces (SEQ ID No. 32);

FIG. 38 shows a polypeptide sequence of a lipid acyltransferase enzyme from Streptomyces (SEQ ID No. 33);

FIG. 39 shows a polypeptide sequence of a lipid acyltransferase enzyme from Termobifida (SEQ ID No. 34);

FIG. 40 shows a nucleotide sequence encoding a lipid acyltransferase enzyme from Termobifida_(SEQ ID No. 35);

FIG. 41 shows a polypeptide sequence of a lipid acyltransferase enzyme from Termobifida (SEQ ID No. 36);

FIG. 42 shows a polypeptide of a lipid acyltransferase enzyme from Corynebacterium\effciens\GDSx 300 aa_(SEQ ID No. 37);

FIG. 43 shows a nucleotide sequence encoding a lipid acyltransferase enzyme from Corynebacterium\effciens\GDSx 300 aa_(SEQ ID No. 38);

FIG. 44 shows a polypeptide of a lipid acyltransferase enzyme from Novosphingobium\aromaticivorans\GDSx 284 aa_(SEQ ID No. 39);

FIG. 45 shows a nucleotide sequence encoding a lipid acyltransferase enzyme from Novosphingobium\aromaticivorans\GDSx 284 aa (SEQ ID No. 40);

FIG. 46 shows a polypeptide of a lipid acyltransferase enzyme from Streptomyces coelicolor\GDSx 268 aa (SEQ ID No. 41);

FIG. 47 shows a nucleotide sequence encoding a lipid acyltransferase enzyme from Streptomyces coelicolor\GDSx 268 aa (SEQ ID No. 42);

FIG. 48 shows a polypeptide of a lipid acyltransferase enzyme from Streptomyces averitilis\GDSx 269 aa (SEQ ID No. 43);

FIG. 49 shows a nucleotide sequence encoding a lipid acyltransferase enzyme from Streptomyces avermitilis\GDSx 269 aa (SEQ ID No. 44);

FIG. 50 shows a polypeptide of a lipid acyltransferase enzyme from Streptomyces (SEQ ID No. 45);

FIG. 51 shows a nucleotide sequence encoding a lipid acyltransferase enzyme from Streptomyces (SEQ ID No. 46);

FIG. 52 shows a ribbon representation of the 1IVN.PDB crystal structure which has glycerol in the active site. The Figure was made using the Deep View Swiss-PDB viewer;

FIG. 53 shows 1IVN.PDB Crystal Structure—Side View using Deep View Swiss-PDB viewer, with glycerol in active site—residues within 10 Å of active site glycerol are coloured black;

FIG. 54 shows 1IVN.PDB Crystal Structure—Top View using Deep View Swiss-PDB viewer, with glycerol in active site—residues within 10 Å of active site glycerol are coloured black;

FIG. 55 shows alignment 1;

FIG. 56 shows alignment 2;

FIGS. 57 and 58 show a alignments of 1IVN to P10480 (P10480 is the database sequence for A. hydrophila enzyme), this alignment was obtained from the PFAM database and used in the model building process;

FIG. 59 shows an alignment where P10480 is the database sequence for Aeromonas hydrophila. This sequence is used for the model construction and the site selection. Note that the full protein is depicted, the mature protein (equivalent to SEQ ID No. 2) starts at residue 19. A. sal is Aeromonas salmonicida (SEQ ID No. 28) GDSX lipase, A. hyd is Aeromonas hydrophila (SEQ ID No. 26) GDSX lipase. The consensus sequence contains a * at the position of a difference between the listed sequences;

FIG. 60 shows a typical set of 384 clones, the wild type control lies at the intersection of 0.9PC, 0.8DGDG; and

FIG. 61 shows three areas of interest. Section 1 contains mutants with an increased ratio R but lower activity towards DGDG. Region 2 contains mutants with an increased ratio R and an increased DGDG activity. Region 3 contains clones with an increased PC or DGDG activity, but no increase in the ratio R.

EXAMPLE 1

Modelling of Aeromonas hydrophila GDSx Lipase on 1IVN

The alignment of the Aeromonas hydrophila GDSX lipase amino acid sequence (P10480) to the Escherichia coli Tioesterase amino acid sequence (1IVN) and the Aspergillus aculeatus rhamnogalacturonan acetylesterase amino acid sequence (1DEO) was obtained from the PFAM database in FASTA format. The alignment of P10480 and 1IVN was fed into an automated 3D structure modeller (SWISS-MODELLER server at www.expasy.org) together with the 1IVN.PDB crystal structure coordinates file FIG. 52). The obtained model for P10480 was structurally aligned to the crystal structures coordinates of 1IVN.PDB and 1DEO.PDB using the ‘Deep View Swiss-PDB viewer’ (obtained from www.expasy.org/spdbv/) (FIG. 53). The amino acid alignment obtained from the PFAM database (alignment 1—(FIG. 55)) was modified based on the structural alignment of 1DEO.PDB and 1IVN.PDB. This alternative amino acid alignment is called alignment 2 (FIG. 56).

The 1IVN.PDB structure contains a glycerol molecule. This molecule is considered to be in the active site it is in the vicinity of the catalytic residues. Therefore, a selection can be made of residues that are close to the active site which, due to their vicinity, are likely to have an influence on substrate binding, product release, and/or catalysis. In the 1IVN.PDB structure, all amino acids within a 10 Å sphere centered on the central carbon atom of the glycerol molecule in the active site were selected (amino acid set 1) (See FIG. 53 and FIG. 54).

The following amino acids were selected from the P10480 sequence; (1) all amino acids in P10480 corresponding to the amino acid set 1 in alignment 1; (2) all amino acids in P10480 corresponding to the amino acid set 1 in alignment 2; (3) from the overlay of the P10480 model and 1IVN all amino acids in the P10480 model within 12 Å from the glycerol molecule in 1IVN. All three groups combined give amino acid set 2.

Sequence P10480 was aligned to “AAG09804.1 GI:9964017 glycerophospholipid-cholesterol acyltransferase [Aeromonas salmonicida]” and the residues in AAG09804 corresponding to amino acid set 2 were selected in amino acid set 3.

Set 1, 2, and 3

Amino Acid Set 1 (Note That These are Amino Acids in 1IVN—FIG. 57 and FIG. 58.)

Gly8, Asp9, Ser10, Leu11, Ser12, Tyr15, Gly44, Asp45, Thr46, Glu69, Leu70, Gly71, Gly72, Asn73, Asp74, Gly75, Leu76, Gln106, Ile107, Arg108, Leu109, Pro110, Tyr113, Phe121, Phe139, Phe140, Met141, Tyr145, Met151, Asp154, Gly155, Ile156, His157, Pro158

The highly conserved motifs, such as GDSx and catalytic residues, were deselected from set 1 (residues underlined).

Amino Acid Set 2 (Note That the Numbering of the Amino Acids Refers to the Amino Acids in the P10480 Mature Sequence)

Leu17, Lys22, Met23, Gly40, Asn80, Pro81, Lys82, Asn87, Asn88, Trp111, Val112, Ala114, Tyr117, Leu118, Pro156, Gly159, Gln160, Asn161, Pro162, Ser163, Ala164, Arg165, Ser166, Gln167, Lys168, Val169, Val170, Glu171, Ala172, Tyr179, His180, Asn181, Met209, Leu210, Arg211, Asn215, Lys284, Met285, Gln289, Val290

Amino acid set 3 is identical to set 2 but refers to the Aeromonas salmonicida (SEQ ID No. 28) mature sequence, i.e. the amino acid residue numbers are 18 higher in set 3 as this reflects the difference between the amino acid numbering in the mature protein (SEQ ID No. 2) compared with the protein including a signal sequence (SEQ ID No. 28).

The mature proteins of Aeromonas salmonicida GDSX (SEQ ID No. 28) and Aeromonas hydrophila GDSX (SEQ ID No. 26) differ in five amino acids. These are Thr3Ser, Lys182Gln Glu309Ala, Thr310Asn, Gly318-, where the salmonicida residue is listed first and the hydrophila residue last (FIG. 59). The hydrophila protein is only 317 amino acids long and lacks a residue on position 318. The Aeromonas salmonicidae GDSX has considerably high activity on polar lipids such as galactolipid substrates than the Aeromonas hydrophila protein.

Amino acid set four=Thr3Ser, Lys182Gln Glu309Ala, Thr310Asn, −318Gly

The Alignments 1 and 2 used to obtain the sets

From the crystal structure one can obtain the secondary structure classification. That means, one can classify each amino acid as being part of an alpha-helix or a beta-sheet. FIG. 57 shows the PFAM alignment of 1DEO, 1IVN, and P10480 (the database Aeromonas hydrophila). Added below each line of sequence is the structural classification.

The PFAM database contains alignments of proteins with low sequence identity. Therefore, these alignments are not very good. Although the alignment algorithms (HAMMER profiles) are well suited for recognizing conserved motifs the algorithm is not very good on a detailed level. Therefore it is not surprising to find a disparity between the PFAM alignment and a structural alignment. As a skilled person would be readily aware, one can modify the PFAM alignment based on the structural data. Meaning that one can align those structural elements that overlap.

FIG. 55 shows the original PFAM alignment of 1DEO, 1IVN and P10480. Added to the alignment is the secondary structure information from the crystal structures of 1DEO and 1IVN. Alignment 2 in FIG. 56 shows a manually modified alignment where the match between the secondary structure elements is improved. Based on conserved residues between either 1DEO and P10480 or between 1IVN and P10480 the alignment was modified for P10480 as well. To easily distinguish the sequence blocks the sequence identifiers in alignment 2 have an extra m (1DEOm, 1IVNm, P10480m).

Alignment 3 is a mix of 1 and 2, it gives the alignment per block

EXAMPLE 2 Construction of Site Scan Libraries

The Quick Change Multi Site-Directed Mutagenesis Kit from Stratagene was used according to the manufacturers instruction. For each library a degenerate primer with one NNK or NNS (nucleotide abbreviations) codon was designed. Primer design was performed using the tools available on the Stratagene web site. Primer quality control was further confirmed using standard analysis tools which analyze the primer for the potential of forming hairpins or of forming primer-dimers.

The main concepts of the method are as follows; using a non-strand displacing high-fidelity DNA polymerase such as Pfu-Turbo and a single primer one will linearly amplify the DNA template. This is in contrast to the normal exponential amplification process of a PCR reaction. This linear amplification process ensures a low error frequency. The product is single stranded non-methylated DNA and double stranded hemi-methylated DNA. If the template is obtained from a suitable host organism, then the template is double stranded methylated DNA. This means that the template DNA can be digested with Dpn I endonuclease without digesting the product DNA. Therefore upon transformation of the DNA into a suitable host only a very low frequency of the transformants with non-mutagenized plasmid.

EXAMPLE 3 Selection of Winners from a Site Scan Library

Two alternative approaches are described; library sequencing followed by analysis of unique amino acids, or library analysis followed by sequencing of the winners.

Selection of winners method 1; library sequencing followed by analysis of unique amino acids.

Site scan libraries were constructed using a degenerate oligo containing one NNK codon, where K stands for G or T and N stands for A, C, G, or T. This means that a set of clones constructed from an amplification reaction using an NNK primer (also known as ‘a site scan library’) contains in principle 32 unique codons (4×4×2=32 combination options). Assuming no bias due, the number of clones that one needs to pick to have a 95% chance of picking every one of the 32 codons at least once is 95. This can be calculated using the following formula n={log(1−c)}/{log(1−f)}  Formula 1;

Where n is the number of clones, c is the fraction value of the confidence interval, for example the 95% confidence interval has a value of 0.95 and the 99% confidence interval has a fraction value of 0.99, and f is the frequency with which each individual codon occurs, which for an NNK primer is 1/32 or 0.03125. Solving the formula for n gives 94.36 or 95 clones. If a 95% confidence interval is deemed to be too low, or if one is unable to avoid bias in one or more steps of the library construction process, one can decide to assay or sequence more clones. For example, in formula 1, if n is set to 384, f to 1/32 or 0.03125 then the confidence interval c is much larger than 99%. Even if 60% of the clones contain the same mutation or the wild type codon, then 363 clones will give a 99% confidence of obtaining all 32 codons. From this one can conclude that, 384 clones will have a 99% confidence of containing each of the 32 codons at least once.

A colony PCR was performed (a PCR reaction on a bacterial colony or on a bacterial liquid culture to amplify a fragment from a plasmid inside a bacterium, and subsequently sequencing that part of the fragment which has been mutagenised is an established procedure. Colony PCR can be routinely performed for sets of 96 due to the availability of prefabricated material (also known as kits) for colony PCR, sequencing, and sequence purification. This entire procedure is offered as a service by several commercial companies such as AGOWA GmbH, Glienicker weg 185, D-12489 Berlin, Germany.

After analysing the 96 sequence reactions, the individual clones were selected representing one for each codon that is available in the set of 96 sequences. Subsequently, the individual clones were grown and the recombinant protein expressed. The unit activity per quantity of protein in the assays described in Example 4 was performed.

Selection of winners method 2; library screening followed by sequencing of the winners

Although one could choose to sequence 384 clones, one may also assay them and select improved variants before sequencing.

A number of issues should be considered when such a number of samples are screened. Without being exhaustive, although it is possible to select variants with altered activity on one substrate, the difference in expression level between 384 cultures can be substantial even if one uses a 384 well microtiter plate, resulting in a high background. Therefore, measuring two activities and selecting winners based on a change in ratio is a preferred method. To illustrate, if two activities have a certain ratio R then regardless of the absolute amount of enzyme present, the ratio between the two activities will always be R. A change in the R value indicates a mutation that changed one activity relative to the second activity.

FIG. 60 shows a data set obtained from the site scan library. The clones are all tested for activity towards phosphatidyl choline (PC) and digalactosyl diglyceride (DGDG). All clones, which can be mutated or not, that exhibit no change in the R value will lie on a straight line with a certain margin of error. Disregarding these clones three groups of interest appear in FIG. 61.

Section 1 in FIG. 61 contains all the clones that have a significantly higher R than the wild-type (not mutated) but lower overall DGDG activity. Section 2 contains those clones that have both a higher R value and a higher DGDG activity than the wild type. Section 3 contains clones that do not have a higher R value, but that do have a significantly higher DGDG or PC activity.

If one is interested in variants with an increased activity towards DGDG then section 2 contains the most interesting variants and section 3 contains variants of interest as well. The variants in Section 3 which show a large increase in hydrolytic activity may be accompanied by a decrease in transferase activity.

One thing is worth noticing, if a specificity determining residue is hit, most of the 20 possible amino acids could yield a very different R value. However, if the library contains a large bias towards a single amino acid (for example 60% is Tyrosine) then all those variants will still lie on a straight line.

EXAMPLE 4 Assays for PC and DGDG Activity in a 384 Well Microtiter Plate

Start Material

-   -   EM media     -   Plate with transformants     -   Plate with wild type     -   384 plates     -   colony picker     -   Waco NEFA-C kit     -   PC and DGDG solutions in a 384 plate

Part 1—Picking Colonies

-   -   Pick colonies into a 384 plate filled with EM medium     -   Skip 4 wells and inoculate those with colonies containing the         non-mutated backbone     -   Grow o/n at 30° C., 200 rpm shaking speed

Part 2—Incubation on Substrate

-   -   Centrifuge the o/n grown plates; 2500 rpm, 20 min     -   Transfer 10 μl supernatant from each well to 2 empty 384 plates     -   Add 5 μl 12.5 mM DGDG to one of the plates, add 5 μl 12.5 mM PC         to the other plate     -   Incubate both plates 2 hrs at 37° C., shake at start to mix then         stop the shaking     -   Continue with the NEFA C procedure

Part 3—NEFA-C Procedure

-   -   Add 10 μl A solution     -   Incubate 10 min 37° C., 300 rpm     -   Add 20 μl B solution     -   Incubate 10 min 37° C., 300 rpm     -   Read the plate at 550 nm

Substrate Composition—in mM

-   -   25 mM PC eller DGDG     -   10 mM CaCl₂     -   60 mM Triton X 100     -   15 mM NaN₃     -   20 mM Briton Robinson pH 5.0

EXAMPLE 5 Selected Variants

Determination of Enzyme Activity

To determine the enzymatic activity towards various substrates 4 μl enzyme solution was incubated with 11 μl substrate for 60 minutes at 37° C. Subsequently the amount of free fatty acids was determined using the WACO NEFA-C kit. To the 15 μl enzyme+substrate mix 75 μl NEFA solution A was added and incubated for 15 minutes at 37° C. Subsequently 150 μl NEFA solution B was added and incubated for 15 minutes. Subsequently the optical density (OD) of the sample was measured at 550 nm.

As a control, from each variant 4 μl enzyme solution was incubated with 11 μl HEPES buffer for 60 min at 37° C. Subsequently the amount of free fatty acids was determined as described above. The OD values of this control sample was deducted from the observed OD on each substrate to obtain a corrected activity.

Four different substrates were used, the composition was in general 30 mg lipid, 4.75 ml 50 mM HEPES buffer pH 7, 42.5 μl 0.6 M CaCl2, 200 μl 10% Triton X-100 H2O2-free. The 30 mg lipid was either phosphatidyl choline (PC), PC with cholsterol in a 9 to 1 ratio, digalactosyl diglyceride (DGDG), or DGDG with cholesterol in a 9 to 1 ratio.

Selection of Improved Variants

Variants with Improved Activity Towards PC

Those variants that showed an increase in the OD relative to the wild type enzyme when incubated on PC were selected as variants with improved phospholipase activity.

Variants with Improved Activity Towards DGDG

Those variants that showed an increase in the OD relative to the wild type enzyme when incubated on DGDG were selected as variants with improved activity towards DGDG.

Variants with Improved Specificity Towards DGDG

The specificity towards DGDG is the ratio between the activity towards DGDG and the activity towards phosphatidylcholine (PC). Those variants that showed a higher ratio between DGDG and PC than the wild type were selected as variants with improved specificity towards DGDG.

Variants with Improved Transferase Activity with PC as the Acyl Donor

The difference in the amount of free fatty acids formed when one incubates an enzyme on PC and on PC with cholesterol is an indication of the amount of transferase activity relative to the amount of hydrolytic activity. Transferase activity will not cause the formation of free fatty acids. The transferase preference is the ratio between the free fatty acids formed when PC is used as a substrate and the free fatty acids formed when PC with cholesterol is used as a substrate. Those variants that show an increase in the transferase preference and show a higher than wild type activity towards PC were selected as having improved transferase activity.

Variants with Improved Transferase Activity with DGDG as the Acyl Donor

The difference in the amount of free fatty acids formed when one incubates an enzyme on DGDG and on DGDG with cholesterol is an indication of the amount of transferase activity relative to the amount of hydrolytic activity. Transferase activity will not cause the formation of free fatty acids. The transferase preference is the ratio between the free fatty acids formed when DGDG is used as a substrate and the free fatty acids formed when DGDG with cholesterol is used as a substrate. Those variants that show an increase in the transferase preference and show a higher than wild type activity towards DGDG were selected as having improved transferase activity.

Selected Variants

For each of the four selection criteria above a number of variants were selected.

The “wild type” enzyme in this example is A. salmonicida (SEQ ID No. 28).

Variants with Improved Activity Towards PC: PC Thr3Asn 158.0 Thr3Gln 151.5 Thr3Lys 141.5 Thr3Arg 133.0 Glu309Ala 106.0 Thr3Pro 101.5 Thr3Met 96.0 wild-type 86.5

Variants with Improved Activity Towards DGDG: DGDG Lys182Asp 66.5 Glu309Ala 60 Tyr230Thr 59 Tyr230Gly 57.5 Tyr230Gly 51 Thr3Gln 44.5 wild-type 43.5

Variants with Improved Specificity Towards DGDG: R _(DGDG/PC) PC DGDG Lys182Asp 1.02 65.5 66.5 Tyr230Gly 0.79 72.5 57.5 Tyr230Gly 0.78 65.0 51.0 Tyr230Thr 0.75 78.5 59.0 Tyr230Val 0.71 58.0 41.0 Asp157Cys 0.69 48.0 33.0 Glu309Pro 0.58 73.5 42.5 Glu309Ala 0.57 106.0 60.0 Gly318Ile 0.53 69.5 36.5 Tyr230Arg 0.50 63.5 32.0 Tyr230Met 0.50 64.5 32.5 wild-type 0.50 86.5 43.5

Variants with Improved Transferase Activity with PC as the Acyl Donor: R_(PC+Cho/PC) PC PC + Cho Thr3Lys 0.54 142 76 Thr3Arg 0.55 133 73 Thr3Gln 0.63 152 96 Thr3Asn 0.64 158 101 Thr3Pro 0.67 102 68 Thr3Met 0.78 96 75 wild-type 0.83 87 72

Variants with Improved Transferase Activity with DGDG as the Acyl Donor: R_(DGDG+Cho/DG) _(DG) DGDG Tyr230Thr 1.10 59 Lys182Asp 1.39 67 Tyr230Gly 1.55 58 Glu309Ala 1.78 60 wild-type 1.78 44

EXAMPLE 6 Transferase Assay Phospholipid:Cholesterol

Phospholipid can be replaced by DGDG to provide a transferase assay from a galacolipid. Other acceptors for example, glycerol, glucose, hydroxy acids, proteins or maltose can also be used in the same assay.

300 mg Phosphatidylcholine (Avanti #441601):Cholesterol(Sigma C8503) 9:1 is scaled in a Wheaton glass. 10 ml 50 mM HEPES buffer pH 7.0 is added and stirring at 40° C. disperses the substrate

0.5 ml substrate is transferred to a 4 ml vial and placed in a heating block at 40° C. 0.050 ml transferase solution is added, also a control with 0.050 ml water is analysed in the same way. The reaction mixture is agitated for 4 hours at 40° C. The sample is then frozen and lyophilised and analysed by GLC.

Calculation:

From the GLC analysis the content of free fatty acids and cholesterol ester is calculated.

The enzymatic activity is calculated as: ${\%\quad{Transferase}\quad{activity}} = {{{\frac{\Delta\quad\%\quad{cholesterol}\quad{{ester}/\left( {{Mv}\quad{sterol}\quad{ester}} \right)} \times 100}{\begin{matrix} {{\Delta\quad\%\quad{cholesterol}\quad{{ester}/\left( {{Mv}\quad{cholesterol}\quad{ester}} \right)}} +} \\ {\Delta\quad\%\quad{fatty}\quad{{acid}/\left( {{Mv}\quad{fatty}\quad{acid}} \right)}} \end{matrix}}.\%}\quad{Hydrolyse}\quad{activity}} = {\frac{\Delta\quad\%\quad{fatty}\quad{{acid}/\left( {{Mv}\quad{fatty}\quad{acid}} \right)} \times 100}{\begin{matrix} {{\Delta\quad\%\quad{cholesterol}\quad{{ester}/\left( {{Mv}\quad{cholesterol}\quad{ester}} \right)}} +} \\ {\Delta\quad\%\quad{fatty}\quad{{acid}/\left( {{Mv}\quad{fatty}\quad{acid}} \right)}} \end{matrix}}.}}$  Ratio Transferase/Hydrolyse=% transferase activity/% Hydrolyse activity Where: Δ % cholesterol ester=% cholesterol ester(sample)−% cholesterol ester(control). Δ % fatty acid=% fatty acid(sample)−% fatty acid(control).

Transferase Assay Galactolipid:Cholesterol.

300 mg Digalactosyldiglyceride (>95%, from Wheat lipid):Cholesterol(Sigma) 9:1 is scaled in a Wheaton glass. 10 ml 50 mM HEPES buffer pH 7.0 is added and stirring at 40° C. disperses the substrate.

0.5 ml substrate is transferred to a 4 ml vial and placed in a heating block at 40° C. 0.050 ml transferase solution is added, also a control with 0.050 ml water is analysed in the same way. The reaction mixture is agitated for 4 hours at 40° C. The sample is then frozen and lyophilised and analysed by GLC.

Calculation:

From the GLC analysis the content of free fatty acids and cholesterol ester is calculated.

The enzymatic activity is calculated as: ${\%\quad{Transferase}\quad{activity}} = {{{\frac{\Delta\quad\%\quad{cholesterol}\quad{{ester}/\left( {{Mv}\quad{sterol}\quad{ester}} \right)} \times 100}{\begin{matrix} {{\Delta\quad\%\quad{cholesterol}\quad{{ester}/\left( {{Mv}\quad{cholesterol}\quad{ester}} \right)}} +} \\ {\Delta\quad\%\quad{fatty}\quad{{acid}/\left( {{Mv}\quad{fatty}\quad{acid}} \right)}} \end{matrix}}.\%}\quad{Hydrolyse}\quad{activity}} = {\frac{\Delta\quad\%\quad{fatty}\quad{{acid}/\left( {{Mv}\quad{fatty}\quad{acid}} \right)} \times 100}{\begin{matrix} {{\Delta\quad\%\quad{cholesterol}\quad{{ester}/\left( {{Mv}\quad{cholesterol}\quad{ester}} \right)}} +} \\ {\Delta\quad\%\quad{fatty}\quad{{acid}/\left( {{Mv}\quad{fatty}\quad{acid}} \right)}} \end{matrix}}.}}$  Ratio Transferase/Hydrolyse=% transferase activity/% Hydrolyse activity Where: Δ % cholesterol ester=% cholesterol ester(sample)−% cholesterol ester(control). Δ % fatty acid=% fatty acid(sample)−% fatty acid(control)

EXAMPLE 7 Variants of a Lipid Acyltransferase for Aeromonas hydrophila (SEQ ID No. 26)

Mutations were introduced using the QuikChange™ Multi-Site Directed Mutagenesis kit from Stratagene, La Jolla, Calif. 92037, USA following the instructions provided by Stratagene.

Variants at Tyr256 showed an increased activity towards phospholipids.

Variants at Tyr256 and Tyr260 showed an increased activity towards galactolipids.

Variants at Tyr265 showed an increased transferase activity with galactolipids as the acyl donor.

The numbers indicate positions on the following sequence: An enzyme from Aeromonas hydrophila the amino acid sequence of which is shown as SEQ ID No. 26. The nucleotide sequence is as shown as SEQ ID No. 27.

The invention will now be further described by the following numbered paragraphs:

1. A method of producing a variant lipid acyltransferase enzyme comprising: (a) selecting a parent enzyme which is a lipid acyltransferase enzyme characterised in that the enzyme comprises the amino acid sequence motif GDSX, wherein X is one or more of the following amino acid residues L, A, V, I, F, Y, H, Q, T N, M or S; (b) modifying one or more amino acids to produce a variant lipid acyltransferase; (c) testing the variant lipid acyltransferase for activity on a galactolipid substrate, and optionally a phospholipid substrate and/or optionally a triglyceride substrate; (d) selecting a variant enzyme with an enhanced activity towards galactolipids compared with the parent enzyme; and optionally (e) preparing a quantity of the variant enzyme.

2. A method according to paragraph 1 wherein one or more of the one or more of the following amino acid residues identified by alignment with SEQ ID No. 2 is modified compared with a parent sequence SEQ ID No. 2: Ser3, Leu17, Ala114, Trp111, Tyr117, Leu118, Pro156, Gly159, Gln160, Asn161, Pro162, Ser163, Ala164, Arg165, Ser166, Gln167, Lys168, Val169, Val170, Glu171, Ala172, Tyr179, Gln182, Lys187, His180, Asn181, Met209, Leu210, Arg211, Asn215, Lys284, Met285, Gln289, Val290, Ala309, Ser310, Lys22, Met23, Gly40, Asn80, Pro81, Lys82, Val112, Asn87, Asn88, −318.

3. A method according to paragraph 1 or paragraph 2 wherein the parent enzyme comprises an amino acid sequence as shown as SEQ ID No. 2, SEQ ID No. 3, SEQ ID No. 4, SEQ ID No. 5, SEQ ID No. 6, SEQ ID No. 12, SEQ ID No. 14, SEQ ID No. 16, SEQ ID No. 18, SEQ ID No. 20, SEQ ID No. 22, SEQ ID No. 24, SEQ ID No. 26, SEQ ID No. 28, SEQ ID No. 30, SEQ ID No. 33, SEQ ID No. 34, SEQ ID No. 36, SEQ ID No. 37, SEQ ID No. 39, SEQ ID No. 41, SEQ ID No. 43 or SEQ ID No. 45, or an amino acid sequence which has at least 70% identity therewith.

4. A method according to any one of paragraphs 1-3 wherein amino acid residue 18 of the parent sequence identified by alignment with SEQ ID No. 2 is substituted by one of the following amino acids A, L, M, F, W, K, Q, E, P, I, C, Y, H, R, N, D, T.

5. A method according to any one of the preceding paragraphs wherein amino acid residue 30 of the parent sequence identified by alignment with SEQ ID No. 2 is by one of the following amino acids A, G, L, M, W, K, Q, S, E, P, V, I, C, H, R, N, D, T.

6. A method according to any one of the preceding paragraphs wherein amino acid residue 20 of the parent sequence identified by alignment with SEQ ID No. 2 is by one of the following amino acids A, G, L, M, W, K, Q, S, E, P, V, I, C, H, R, N, D, T.

7. A method according to any one of the preceding paragraphs wherein the parent enzyme is an enzyme which comprises the amino acid sequence shown as SEQ ID No. 2 and/or SEQ ID No. 28.

8. A method according to any one of the preceding paragraphs wherein Preferably, the X of the GDSX motif is L.

9. A method according to any one of the preceding paragraphs wherein the method further comprises one or more of the following steps: structural homology mapping or sequence homology alignment.

10. A method according to paragraph 9 wherein the structural homology mapping comprises one or more of the following steps:

-   -   a) aligning a parent sequence with a structural model (1IVN.PDB)         shown in FIG. 52;     -   b) selecting one or more amino acid residue within a 10 Å sphere         centred on the central carbon atom of the glycerol molecule in         the active site (see FIG. 53); and     -   c) modifying one or more amino acids selected in accordance with         step (b) in said parent sequence.

11. A method according to paragraph 9 wherein the structural homology mapping comprises one or more of the following steps:

-   -   a) aligning a parent sequence with a structural model (1IVN.PDB)         shown in FIG. 52;     -   b) selecting one or more amino acids within a 10 Å sphere         centred on the central carbon atom of the glycerol molecule in         the active site (see FIG. 53);     -   c) determining if one or more amino acid residues selected in         accordance with step (b) are highly conserved (particularly are         active site residues and/or part of the GDSx motif and/or part         of the GANDY motif); and     -   d) modifying one or more amino acids selected in accordance with         step (b), excluding conserved regions identified in accordance         with step (c) in said parent sequence.

12. A method according to paragraph 9 wherein the sequence homology alignment comprises one or more of the following steps:

-   -   i) selecting a first parent lipid acyltransferase;     -   identifying a second related lipid acyltransferase having a         desirable activity;     -   aligning said first parent lipid acyltransferase and the second         related lipid acyltransferase;     -   identifying amino acid residues that differ between the two         sequences; and     -   modifying one or more of the amino acid residues identified in         accordance with step (iv) in said parent lipid acyltransferase.

13. A method according to paragraph 9 wherein the sequence homology alignment may comprise one or more of the following steps:

-   -   i) selecting a first parent lipid acyltransferase;     -   ii) identifying a second related lipid acyltransferase having a         desirable activity;     -   iii) aligning said first parent lipid acyltransferase and the         second related lipid acyltransferase;     -   iv) identifying amino acid residues that differ between the two         sequences;     -   v) determining if one or more amino acid residues selected in         accordance with step (iv) are highly conserved (particularly are         active site residues and/or part of the GDSx motif and/or part         of the GANDY motif); and     -   vi) modifying one or more of the amino acid residues identified         in accordance with step (iv) excluding conserved regions         identified in accordance with step (v) in said parent sequence.

14. A method according to any one of the preceding paragraphs wherein one or more of the following modifications is made to the parent enzyme: S3A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; D157A, C, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W or Y; Q182A, C, D, E, F, G, H, I, K, L, M, N, Q, P, R, S, T, V, W, or Y; A309A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W or Y; Y230A, C, D, E, G, H, I, K, L, M, N, P, Q, R, S, T, V or W; a C-terminal addition (−318) of at least one amino acid.

15. A method according to paragraph 14 wherein one or more of the following modifications is made to the parent enzyme S3T, S3N, S3Q, S3K, S3R, S3P, S3M; D157 is substituted with a polar uncharged amino acid, preferably with C, S, T or M, more preferably C; Q182 is substituted with an aliphatic amino acid residue, preferably a polar amino acid, more preferably a polar charged amino acid, more preferably D or E, most preferably D; A309 is substituted with an aliphatic residue, preferably a non-polar residue, preferably G, A, or P, more preferably A; Y230 is substituted with an aliphatic amino acid or one of the following amino acid residues G, D, T, V, R or M, more preferably G, D, T, V, R or M, more preferably G or T; a C-terminal addition comprising one or more of I, L or V.

16. A method according to any one of the preceding paragraphs one or more of the following modifications is made to the parent enzyme K187D, E309A, Y230T, Y230G, S3Q.

17. A method according to any one of the preceding paragraphs wherein one or more of the following modifications is made to the parent enzyme K187D, K187D, Y230G, Y230T, Y230R, Y230M, Y230V, D157C, E309A, G2181.

18. A method according to any one of the preceding paragraphs wherein one or more of the following modifications is made to the parent enzyme S3K, S3R, S3Q, S3N, S3P, S3M.

19. A method according to any one of the preceding paragraphs wherein one or more of the following modifications is made to the parent enzyme Y230T, K187D, Y230G, E309A

20. A variant lipid acyltransferase enzyme characterised in that the enzyme comprises the amino acid sequence motif GDSX, wherein X is one or more of the following amino acid residues L, A, V, I, F, Y, H, Q, T N, M or S, and wherein the variant enzyme comprises one or more amino acid modifications compared with a parent sequence at any one or more of the following amino acid residues when aligned to SEQ ID No. 2: Ser3, Leu17, Ala114, Trp111, Tyr117, Leu118, Pro156, Gly159, Gln160, Asn161, Pro162, Ser163, Ala164, Arg165, Ser166, Gln167, Lys168, Val169, Val170, Glu171, Ala172, Tyr179, Gln182, Lys187, His180, Asn181, Met209, Leu210, Arg211, Asn215, Lys284, Met285, Gln289, Val290, Ala309, Ser310, Lys22, Met23, Gly40, Asn80, Pro81, Lys82, Val112, Asn87, Asn88, −318.

21. A variant lipid acyltransferase enzyme according to paragraph 20 wherein the variant enzyme comprises an amino acid sequence, which amino acid sequence is shown as SEQ ID No. 2, SEQ ID No. 3, SEQ ID No. 4, SEQ ID No. 5, SEQ ID No. 6, SEQ ID No. 12, SEQ ID No. 14, SEQ ID No. 16, SEQ ID No. 18, SEQ ID No. 20, SEQ ID No. 22, SEQ ID No. 24, SEQ ID No. 26, SEQ ID No. 28, SEQ ID No. 30, SEQ ID No. 33, SEQ ID No. 34, SEQ ID No. 36, SEQ ID No. 37, SEQ ID No. 39, SEQ ID No. 41, SEQ ID No. 43 or SEQ ID No. 45 except for one or more amino acid modifications at any one or more of the following amino acid residues identified by sequence alignment with SEQ ID No. 2: Ser3, Leu17, Ala114, Trp111, Tyr117, Leu118, Pro156, Gly159, Gln160, Asn161, Pro162, Ser163, Ala164, Arg165, Ser166, Gln167, Lys168, Val169, Val170, Glu171, Ala172, Tyr179, Gln182, Lys187, His180, Asn181, Met209, Leu210, Arg211, Asn215, Lys284, Met285, Gln289, Val290, Ala309, Ser310, Lys22, Met23, Gly40, Asn80, Pro81, Lys82, Val112, Asn87, Asn88, −318.

22. A variant lipid acyltransferase enzyme according to paragraph 20 or paragraph 21 wherein the enzyme comprises one or more of the following amino acid modifications S18A, L, M, F, W, K, Q, E, P, I, C, Y, H, R, N, D, T; Y30A, G, L, M, W, K, Q, S, E, P, V, I, C, H, R, N, D, T; Y230A, G, L, M, W, K, Q, S, E, P, V, I, C, H, R, N, D, T.

23. A variant lipid acyltransferase enzyme according to any one of paragraphs 20-22 wherein the enzyme comprises one or more of the following amino acid modifications: S3A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; D157A, C, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W or Y; Q182A, C, D, E, F, G, H, I, K, L, M, N, Q, P, R, S, T, V, W, or Y; A309A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W or Y; Y230A, C, D, E, G, H, I, K, L, M, N, P, Q, R, S, T, V or W; a C-terminal addition (−318) of at least one amino acid.

24. A variant lipid acyltransferase enzyme according to any one of paragraphs 20-23 wherein the enzyme comprises one or more of the following amino acid modifications: S3T, S3N, S3Q, S3K, S3R, S3P, S3M; D157 is substituted with a polar uncharged amino acid, preferably with C, S, T or M, more preferably C; Q182 is substituted with an aliphatic amino acid residue, preferably a polar amino acid, more preferably a polar charged amino acid, more preferably D or E, most preferably D; A309 is substituted with an aliphatic residue, preferably a non-polar residue, preferably G, A, or P, more preferably A; Y230 is substituted with an aliphatic amino acid or one of the following amino acid residues G, D, T, V, R or M, more preferably G, D, T, V, R or M, more preferably G or T; a C-terminal addition comprising one or more of I, L or V.

25. A variant lipid acyltransferase enzyme according to any one of paragraphs 20-24 wherein the enzyme comprises one or more of the following amino acid modifications: K187D, E309A, Y230T, Y230G, S3Q.

26. A variant lipid acyltransferase enzyme according to any one of paragraphs 20-25 wherein the enzyme comprises one or more of the following amino acid modifications: K187D, K187D, Y230G, Y230T, Y230R, Y230M, Y230V, D157C, E309A, G2181.

27. A variant lipid acyltransferase enzyme according to any one of paragraphs 20-26 wherein the enzyme comprises one or more of the following amino acid modifications: S3K, S3R, S3Q, S3N, S3P, S3M.

28. A variant lipid acyltransferase enzyme according to any one of paragraphs 20-27 wherein the enzyme comprises one or more of the following amino acid modifications: Y230T, K187D, Y230G, E309A.

29. A variant lipid acyltransferase enzyme according to any one paragraphs 20-28 wherein the variant enzyme has an enhanced ratio of activity on galactolipids to either phospholipids and/or triglycerides when compared with the parent enzyme.

30. A variant lipid acyltransferase enzyme according to any one of paragraphs 20-29 wherein the variant enzyme is an enzyme which comprises an amino acid sequence, which amino acid sequence is shown as SEQ ID No. 2 or SEQ ID No. 28 except for one or more amino acid modifications at any one or more of the following amino acid residues: Ser3, Leu17, Ala114, Trp111, Tyr117, Leu118, Pro156, Gly159, Gln160, Asn161, Pro162, Ser163, Ala164, Arg165, Ser166, Gln167, Lys168, Val169, Val170, Glu171, Ala172, Tyr179, Gln182, Lys187, His180, Asn181, Met209, Leu210, Arg211, Asn215, Lys284, Met285, Gln289, Val290, Ala309, Ser310, Lys22, Met23, Gly40, Asn80, Pro81, Lys82, Val112, Asn87, Asn88.

31. Use of a variant lipolytic enzyme according to any one of paragraphs 20-30 or obtained by the method according to any one of paragraphs 1-19 in a substrate for preparing a lyso-glycolipid, for example digalactosyl monoglyceride (DGMG) or monogalactosyl monoglyceride (MGMG) by treatment of a glycolipid (e.g. digalactosyl diglyceride (DGDG) or monogalactosyl diglyceride (MGDG)) with the variant lipolytic enzyme according to the present invention or obtained by a method according to the present invention to produce the partial hydrolysis product, i.e. the lyso-glycolipid.

32. Use according to paragraph 31 wherein the substrate is a foodstuff.

33. A method of preparing a foodstuff the method comprising adding a variant lipolytic enzyme according to any one of paragraphs 20-30 or obtained by the method according to any one of paragraphs 1-19 to one or more ingredients of the foodstuff.

34. A method of preparing a baked product from a dough, the method comprising adding a variant lipolytic enzyme according to any one of paragraphs 20-30 or obtained by the method according to any one of paragraphs 1-19 to the dough.

35. Use of a variant lipolytic enzyme according to any one of paragraphs 20-30 or obtained by the method according to any one of paragraphs 1-19 in a process of treating egg or egg-based products to produce lysophospholipids.

36. A process of enzymatic degumming of vegetable or edible oils, comprising treating the edible or vegetable oil with a variant lipolytic enzyme according to any one of paragraphs 20-30 or obtained by the method according to any one of paragraphs 1-19 so as to hydrolyse a major part of the polar lipids (e.g. phospholipid and/or glycolipid).

37. Use of a variant lipolytic enzyme according to any one of paragraphs 20-30 or obtained by the method according to any one of paragraphs 1-19 in a process for reducing the content of a phospholipid in an edible oil, comprising treating the oil with said variant lipolytic enzyme so as to hydrolyse a major part of the phospholipid, and separating an aqueous phase containing the hydrolysed phospholipid from the oil.

38. Use of a variant lipolytic enzyme according to any one of paragraphs 20-30 or obtained by the method according to any one of paragraphs 1-19 in the bioconversion of polar lipids (preferably glycolipids) to make high value products, such as carbohydrate esters and/or protein esters and/or protein subunit esters and/or a hydroxy acid ester.

39. An immobilised variant lipolytic enzyme according to any one of paragraphs 20-30 or obtained by the method according to any one of paragraphs 1-19.

40. A variant lipolytic enzyme generally as described herein with reference to the figures and examples.

All publications mentioned in the above specification are herein incorporated by reference. Various modifications and variations of the described methods and system of the present invention will be apparent to those skilled in the art without departing from the scope and spirit of the present invention. Although the present invention has been described in connection with specific preferred embodiments, it should be understood that the invention as claimed should not be unduly limited to such specific embodiments. Indeed, various modifications of the described modes for carrying out the invention which are obvious to those skilled in biochemistry and biotechnology or related fields are intended to be within the scope of the following claims. 

1. A method of producing a variant lipid acyltransferase enzyme comprising: (a) selecting a parent enzyme which is a lipid acyltransferase enzyme characterised in that the enzyme comprises the amino acid sequence motif GDSX, wherein X is one or more of the following amino acid residues L, A, V, I, F, Y, H, Q, T N, M or S; (b) modifying one or more amino acids to produce a variant lipid acyltransferase; (c) testing the variant lipid acyltransferase for activity on a galactolipid substrate, and optionally a phospholipid substrate and/or optionally a triglyceride substrate; (d) selecting a variant enzyme with an enhanced activity towards galactolipids compared with the parent enzyme; and optionally (e) preparing a quantity of the variant enzyme.
 2. The method according to claim 1 wherein one or more of the one or more of the following amino acid residues identified by alignment with SEQ ID No. 2 is modified compared with a parent sequence SEQ ID No. 2: Ser3, Leu17, Ala114, Trp111, Tyr117, Leu118, Pro156, Gly159, Gln160, Asn161, Pro162, Ser163, Ala164, Arg165, Ser166, Gln167, Lys168, Val169, Val170, Glu171, Ala172, Tyr179, Gln182, Lys187, His180, Asn181, Met209, Arg211, Asn215, Lys284, Met285, Gln289, Val290, Ala309, Ser310, Lys22, Met23, Gly40, Asn80, Pro81, Lys82, Val112, Asn87, Asn88, −318.
 3. The method according to claim 1 wherein the parent enzyme comprises an amino acid sequence as shown as SEQ ID No. 2, SEQ ID No. 3, SEQ ID No. 4, SEQ ID No. 5, SEQ ID No. 6, SEQ ID No. 12, SEQ ID No. 14, SEQ ID No. 16, SEQ ID No. 18, SEQ ID No. 20, SEQ ID No. 22, SEQ ID No. 24, SEQ ID No. 26, SEQ ID No. 28, SEQ ID No. 30, SEQ ID No. 33, SEQ ID No. 34, SEQ ID No. 36, SEQ ID No. 37, SEQ ID No. 39, SEQ ID No. 41, SEQ ID No. 43 or SEQ ID No. 45, or an amino acid sequence which has at least 70% identity therewith.
 4. The method according to claim 1 wherein amino acid residue 18 of the parent sequence identified by alignment with SEQ ID No. 2 is substituted by one of the following amino acids A, L, M, F, W, K, Q, E, P, I, C, Y, H, R, N, D, T.
 5. The method according to claim 1 wherein amino acid residue 30 of the parent sequence identified by alignment with SEQ ID No. 2 is by one of the following amino acids A, G, L, M, W, K, Q, S, E, P, V, I, C, H, R, N, D, T.
 6. The method according to claim 1 wherein amino acid residue 20 of the parent sequence identified by alignment with SEQ ID No. 2 is by one of the following amino acids A, G, L, M, W, K, Q, S, E, P, V, I, C, H, R, N, D, T.
 7. The method according to claim 1 wherein the parent enzyme is an enzyme which comprises the amino acid sequence shown as SEQ ID No. 2 and/or SEQ ID No.
 28. 8. The method according to claim 1 wherein the X of the GDSX motif is L.
 9. The method according to claim 1 wherein the method further comprises one or more of the following steps: structural homology mapping or sequence homology alignment.
 10. The method according to claim 9 wherein the structural homology mapping comprises one or more of the following steps: a) aligning a parent sequence with a structural model (1IVN.PDB) shown in FIG. 52; b) selecting one or more amino acid residue within a 10 Å sphere centred on the central carbon atom of the glycerol molecule in the active site (see FIG. 53); and c) modifying one or more amino acids selected in accordance with step (b) in said parent sequence.
 11. The method according to claim 9 wherein the structural homology mapping comprises one or more of the following steps: a) aligning a parent sequence with a structural model (1IVN.PDB) shown in FIG. 52; b) selecting one or more amino acids within a 10 Å sphere centred on the central carbon atom of the glycerol molecule in the active site (see FIG. 53); c) determining if one or more amino acid residues selected in accordance with step (b) are highly conserved (particularly are active site residues and/or part of the GDSx motif and/or part of the GANDY motif, SEQ ID NO: 54); and d) modifying one or more amino acids selected in accordance with step (b), excluding conserved regions identified in accordance with step (c) in said parent sequence.
 12. The method according to claim 9 wherein the sequence homology alignment comprises one or more of the following steps: a) selecting a first parent lipid acyltransferase; b) identifying a second related lipid acyltransferase having a desirable activity; c) aligning said first parent lipid acyltransferase and the second related lipid acyltransferase; d) identifying amino acid residues that differ between the two sequences; and e) modifying one or more of the amino acid residues identified in accordance with step (iv) in said parent lipid acyltransferase.
 13. The method according to claim 9 wherein the sequence homology alignment may comprise one or more of the following steps: i. selecting a first parent lipid acyltransferase; ii. identifying a second related lipid acyltransferase having a desirable activity; iii. aligning said first parent lipid acyltransferase and the second related lipid acyltransferase; iv. identifying amino acid residues that differ between the two sequences; v. determining if one or more amino acid residues selected in accordance with step (iv) are highly conserved (particularly are active site residues and/or part of the GDSx motif and/or part of the GANDY motif; SEQ ID NO: 54); and vi. modifying one or more of the amino acid residues identified in accordance with step (iv) excluding conserved regions identified in accordance with step (v) in said parent sequence.
 14. The method according to claim 1 wherein one or more of the following modifications is made to the parent enzyme: S3A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; D157A, C, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W or Y; Q182A, C, D, E, F, G, H, I, K, L, M, N, Q, P, R, S, T, V, W, or Y; A309A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W or Y; Y230A, C, D, E, G, H, I, K, L, M, N, P, Q, R, S, T, V or W; a C-terminal addition (−318) of at least one amino acid.
 15. The method according to claim 14 wherein one or more of the following modifications is made to the parent enzyme S3T, S3N, S3Q, S3K, S3R, S3P, S3M; D157 is substituted with a polar uncharged amino acid; Q182 is substituted with an aliphatic amino acid residue; A309 is substituted with an aliphatic residue; Y230 is substituted with an aliphatic amino acid or one of the following amino acid residues G, D, T, V, R or M; a C-terminal addition comprising one or more of I, L or V.
 16. The method according to claim 1 wherein one or more of the following modifications is made to the parent enzyme K187D, E309A, Y230T, Y230G, S3Q.
 17. The method according to claim 1 wherein one or more of the following modifications is made to the parent enzyme K187D, K187D, Y230G, Y230T, Y230R, Y230M, Y230V, D157C, E309A, G2181.
 18. The method according to claim 1 wherein one or more of the following modifications is made to the parent enzyme S3K, S3R, S3Q, S3N, S3P, S3M.
 19. The method according to claim 1 wherein one or more of the following modifications is made to the parent enzyme Y230T, K187D, Y230G, E309A
 20. A variant lipid acyltransferase enzyme characterised in that the enzyme comprises the amino acid sequence motif GDSX, wherein X is one or more of the following amino acid residues L, A, V, I, F, Y, H, Q, T N, M or S, and wherein the variant enzyme comprises one or more amino acid modifications compared with a parent sequence at any one or more of the following amino acid residues when aligned to SEQ ID No. 2: Ser3, Leu17, Ala114, Trp111, Tyr117, Leu118, Pro156, Gly159, Gln160, Asn161, Pro162, Ser163, Ala164, Arg165, Ser166, Gln167, Lys168, Val169, Val170, Glu171, Ala172, Tyr179, Gln182, Lys187, His180, Asn181, Met209, Leu210, Arg211, Asn215, Lys284, Met285, Gln289, Val290, Ala309, Ser310, Lys22, Met23, Gly40, Asn80, Pro81, Lys82, Val112, Asn87, Asn88, −318.
 21. The variant lipid acyltransferase enzyme according to claim 20 wherein the variant enzyme comprises an amino acid sequence, which amino acid sequence is shown as SEQ ID No. 2, SEQ ID No. 3, SEQ ID No. 4, SEQ ID No. 5, SEQ ID No. 6, SEQ ID No. 12, SEQ ID No. 14, SEQ ID No. 16, SEQ ID No. 18, SEQ ID No. 20, SEQ ID No. 22, SEQ ID No. 24, SEQ ID No. 26, SEQ ID No. 28, SEQ ID No. 30, SEQ ID No. 33, SEQ ID No. 34, SEQ ID No. 36, SEQ ID No. 37, SEQ ID No. 39, SEQ ID No. 41, SEQ ID No. 43 or SEQ ID No. 45 except for one or more amino acid modifications at any one or more of the following amino acid residues identified by sequence alignment with SEQ ID No. 2: Ser3, Leu17, Ala114, Trp111, Tyr117, Leu118, Pro156, Gly159, Gln160, Asn161, Pro162, Ser163, Ala164, Arg165, Ser166, Gln167, Lys168, Val169, Val170, Glu171, Ala172, Tyr179, Gln182, Lys187, His180, Asn181, Met209, Leu210, Arg211, Asn215, Lys284, Met285, Gln289, Val290, Ala309, Ser310, Lys22, Met23, Gly40, Asn80, Pro81, Lys82, Val112, Asn87, Asn88, −318.
 22. The variant lipid acyltransferase enzyme according to claim 20 wherein the enzyme comprises one or more of the following amino acid modifications S18A, L, M, F, W, K, Q, E, P, I, C, Y, H, R, N, D, T; Y30A, G, L, M, W, K, Q, S, E, P, V, I, C, H, R, N, D, T; Y230A, G, L, M, W, K, Q, S, E, P, V, I, C, H, R, N, D, T.
 23. The variant lipid acyltransferase enzyme according to claim 20 wherein the enzyme comprises one or more of the following amino acid modifications: S3A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; D157A, C, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W or Y; Q182A, C, D, E, F, G, H, I, K, L, M, N, Q, P, R, S, T, V, W, or Y; A309A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W or Y; Y230A, C, D, E, G, H, I, K, L, M, N, P, Q, R, S, T, V or W; a C-terminal addition (−318) of at least one amino acid.
 24. The variant lipid acyltransferase enzyme according to claim 20 wherein the enzyme comprises one or more of the following amino acid modifications: S3T, S3N, S3Q, S3K, S3R, S3P, S3M; D157 is substituted with a polar uncharged amino acid; Q182 is substituted with an aliphatic amino acid residue; A309 is substituted with an aliphatic residue; Y230 is substituted with an aliphatic amino acid or one of the following amino acid residues G, D, T, V, R or M; a C-terminal addition comprising one or more of I, L or V.
 25. The variant lipid acyltransferase enzyme according to claim 20 wherein the enzyme comprises one or more of the following amino acid modifications: K187D, E309A, Y230T, Y230G, S3Q.
 26. The variant lipid acyltransferase enzyme according to claim 20 wherein the enzyme comprises one or more of the following amino acid modifications: K187D, K187D, Y230G, Y230T, Y230R, Y230M, Y230V, D157C, E309A, G218I.
 27. The variant lipid acyltransferase enzyme according to claim 20 wherein the enzyme comprises one or more of the following amino acid modifications: S3K, S3R, S3Q, S3N, S3P, S3M.
 28. The variant lipid acyltransferase enzyme according to claim 20 wherein the enzyme comprises one or more of the following amino acid modifications: Y230T, K187D, Y230G, E309A.
 29. The variant lipid acyltransferase enzyme according to claim 20 wherein the variant enzyme has an enhanced ratio of activity on galactolipids to either phospholipids and/or triglycerides when compared with the parent enzyme.
 30. The variant lipid acyltransferase enzyme according to claim 20 wherein the variant enzyme is an enzyme which comprises an amino acid sequence, which amino acid sequence is shown as SEQ ID No. 2 or SEQ ID No. 28 except for one or more amino acid modifications at any one or more of the following amino acid residues: Ser3, Leu17, Ala114, Trp111, Tyr117, Leu118, Pro156, Gly159, Gln160, Asn161, Pro162, Ser163, Ala164, Arg165, Ser166, Gln167, Lys168, Val169, Val170, Glu171, Ala172, Tyr179, Gln182, Lys187, His180, Asn181, Met209, Leu210, Arg211, Asn215, Lys284, Met285, Gln289, Val290, Ala309, Ser310, Lys22, Met23, Gly40, Asn80, Pro81, Lys82, Val112, Asn87, Asn88.
 31. A method of using a variant lipolytic enzyme according to claim 20 in a substrate for preparing a lyso-glycolipid, for example digalactosyl monoglyceride (DGMG) or monogalactosyl monoglyceride (MGMG) by treatment of a glycolipid (e.g. digalactosyl diglyceride (DGDG) or monogalactosyl diglyceride (MGDG)) with the variant lipolytic enzyme according to the present invention or obtained by a method according to the present invention to produce the partial hydrolysis product, i.e. the lyso-glycolipid.
 32. A method of using a variant lipolytic enzyme obtained by the method according to claim 1 in a substrate for preparing a lyso-glycolipid, for example digalactosyl monoglyceride (DGMG) or monogalactosyl monoglyceride (MGMG) by treatment of a glycolipid (e.g. digalactosyl diglyceride (DGDG) or monogalactosyl diglyceride (MGDG)) with the variant lipolytic enzyme according to the present invention or obtained by a method according to the present invention to produce the partial hydrolysis product, i.e. the lyso-glycolipid.
 33. The method according to claim 31 wherein the substrate is a foodstuff.
 34. The method according to claim 32 wherein the substrate is a foodstuff.
 35. A method of preparing a foodstuff the method comprising adding a variant lipolytic enzyme according to claim 20 to one or more ingredients of the foodstuff.
 36. A method of preparing a foodstuff the method comprising adding a variant lipolytic enzyme obtained by the method according to claim 1 to one or more ingredients of the foodstuff.
 37. A method of preparing a baked product from a dough, the method comprising adding a variant lipolytic enzyme according to claim 20 to the dough.
 38. A method of preparing a baked product from a dough, the method comprising adding a variant lipolytic enzyme obtained by the method according to claim 1 to the dough.
 39. A method of using a variant lipolytic enzyme according to claim 20 in a process of treating egg or egg-based products to produce lysophospholipids.
 40. A method of using a variant lipolytic enzyme obtained by the method according to claim 1 in a process of treating egg or egg-based products to produce lysophospholipids.
 41. A process of enzymatic degumming of vegetable or edible oils, comprising treating the edible or vegetable oil with a variant lipolytic enzyme according to claim 20 so as to hydrolyse a major part of the polar lipids (e.g. phospholipid and/or glycolipid).
 42. A process of enzymatic degumming of vegetable or edible oils, comprising treating the edible or vegetable oil with a variant lipolytic enzyme obtained by the method according to claim 1 so as to hydrolyse a major part of the polar lipids (e.g. phospholipid and/or glycolipid).
 43. A method of using a variant lipolytic enzyme according to claim 20 in a process for reducing the content of a phospholipid in an edible oil, comprising treating the oil with said variant lipolytic enzyme so as to hydrolyse a major part of the phospholipid, and separating an aqueous phase containing the hydrolysed phospholipid from the oil.
 44. A method of using a variant lipolytic enzyme obtained by the method according to claim 1 in a process for reducing the content of a phospholipid in an edible oil, comprising treating the oil with said variant lipolytic enzyme so as to hydrolyse a major part of the phospholipid, and separating an aqueous phase containing the hydrolysed phospholipid from the oil.
 45. A method of using a variant lipolytic enzyme according to claim 20 in the bioconversion of polar lipids (preferably glycolipids) to make high value products, such as carbohydrate esters and/or protein esters and/or protein subunit esters and/or a hydroxy acid ester.
 46. A method of using a variant lipolytic enzyme obtained by the method according to claim 1 in the bioconversion of polar lipids (preferably glycolipids) to make high value products, such as carbohydrate esters and/or protein esters and/or protein subunit esters and/or a hydroxy acid ester.
 47. An immobilised variant lipolytic enzyme according to claim
 20. 48. An immobilised variant lipolytic enzyme obtained by the method according to claim
 1. 